Methods for Cancer and Immunotherapy Using Prodrugs of Glutamine Analogs

ABSTRACT

and the pharmaceutically acceptable salts thereof, wherein R1, R2, R2′, and X are as defined as set forth in the specification. Compounds having formula (I) are prodrugs that release glutamine analogs, e.g., 6-diazo-5-oxo-L-norleucine (DON).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/885,275, filed on Jan. 31, 2018, which is a continuation inpart of PCT/US2016/044829, filed Jul. 29, 2016, that claims the benefitof U.S. Provisional Application Nos. 62/199,381 and 62/199,566, bothfiled Jul. 31, 2015, the contents of each are incorporated herein byreference in their entirety.

BACKGROUND

Cells under certain conditions may undergo a metabolic switch from ametabolic profile that requires less activity of certain metabolicpathways to meet the cell's energy demands to a metabolic profile thatrequires greater activity of those metabolic pathways or increasedactivity of other metabolic pathways to meet its energy demands. Forexample, cells under certain conditions may undergo a switch towardincreased glycolysis and away from oxidative phosphorylation (OXPHOS).While glycolysis provides less adenosine triphosphate (ATP) thanoxidative phosphorylation, it has been proposed that aerobic glycolysispermits the generation of the substrates necessary for the generation ofamino acids, nucleic acids and lipids, all of which are crucial forproliferation (Vander Heiden et al. (2009) Science 324(5930):1029-1033).This use of glycolysis in the presence of oxygen was first described byOtto Warburg in cancer cells (Warburg (1956) Science 124 (3215):269-270)and was subsequently found to be important in activated T cells (Warburget al. (1958) [Metabolism of leukocytes]. Zeitschrift furNaturforschung. Teil B: Chemie, Biochemie, Biophysik, Biologie 13B(8):515-516). These metabolically reprogrammed cells depend on theincreased activity of certain metabolic pathways, such as pathwaysinvolved in glutamine metabolism, glycolysis, and fatty acid synthesis.However, specific inhibitors of individual enzymes in these metabolicpathways alone have not proven effective because multiple points withineach metabolic pathway are modulated as a cell's metabolism isreprogrammed to meet the extraordinarily large energy demands of theabnormal, harmful, or unhealthy state.

SUMMARY

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning. A Laboratory Manual, 3^(rd) ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. andLane, D., Antibodies—A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988; Freshney, R. I., “Culture of AnimalCells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons,Hoboken, N.J., 2005. Non-limiting information regarding therapeuticagents and human diseases is found in Goodman and Gilman's ThePharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005,Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton& Lange 10^(th) ed. (2006) or 11th edition (July 2009). Non-limitinginformation regarding genes and genetic disorders is found in McKusick,V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes andGenetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12thedition) or the more recent online database: Online MendelianInheritance in Man, OMIM™. McKusick-Nathans Institute of GeneticMedicine, Johns Hopkins University (Baltimore, Md.) and National Centerfor Biotechnology Information, National Library of Medicine (Bethesda,Md.), as of May 1, 2010, World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance inAnimals (OMIA), a database of genes, inherited disorders and traits inanimal species (other than human and mouse), athttp://omia.angis.org.au/contact.shtml.

In some aspects, the presently disclosed subject matter provides amethod for treating a cancer in a subject in need thereof, the methodcomprising: (a) administering a therapeutically effective amount of afirst immunotherapy to the subject, wherein the first immunotherapy is ametabolic reprogramming agent that decreases glutamine metabolicactivity; and (b) optionally administering a therapeutically effectiveamount of a second immunotherapy to the subject.

In particular embodiments, the metabolic reprogramming agent is aglutamine antagonist. In particular embodiments, the metabolicreprogramming agent is a glutamine analog that interferes with aglutamine metabolic pathway. In particular embodiments, the metabolicreprogramming agent is selected from the group consisting of acivicin(L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleaceticacid), azaserine, and 6-diazo-5-oxo-norleucine (DON), and5-diazo-4-oxo-L-norvaline (L-DONV). In particular embodiments, themetabolic reprogramming agent is a prodrug of a glutamine analog thatinterferes with a glutamine metabolic pathway. In particularembodiments, at least one metabolic reprogramming agent is a prodrug ofacivicin, azaserine, DON, and L-DONV. In particular embodiments, atleast one metabolic reprogramming agent is a compound having any one offormula (I), formula (IIA), formula (IIB), or formula (III), below.

In particular embodiments, the method includes simultaneously orsequentially administering a therapeutically effective amount of thesecond immunotherapy, e.g., an immunotherapeutic agent, to the subject.

In particular embodiments, the method includes simultaneously orsequentially administering a therapeutically effective amount of thesecond immunotherapy to the subject, wherein the second immunotherapy isan immune checkpoint blockade therapy. In particular embodiments, theimmune checkpoint blockade therapy is selected from the group consistingof PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, LAG3antagonists, B7-H₃ antagonists, and combinations thereof.

In particular embodiments, the method includes simultaneously orsequentially administering a therapeutically effective amount of thesecond immunotherapy to the subject, wherein the second immunotherapy isan adoptive cellular therapy.

In particular embodiments, the method includes simultaneously orsequentially administering a therapeutically effective amount of thesecond immunotherapy to the subject, wherein the second immunotherapy ismarrow-infiltrating lymphocytes (MILs).

In particular embodiments, the method includes simultaneously orsequentially administering a therapeutically effective amount of thesecond immunotherapy to the subject, wherein the second immunotherapy isan adenosine A2aR blockade.

In particular embodiments, the method includes simultaneously orsequentially administering a therapeutically effective amount of thesecond immunotherapy to the subject, wherein the second immunotherapy isa tumor vaccine.

In particular embodiments, the method includes simultaneously orsequentially administering a therapeutically effective amount of thesecond immunotherapy to the subject, wherein the second immunotherapy isa passive immunotherapy antibody. In particular embodiments, the passiveimmunotherapy antibody is selected from the group consisting ofbevacizumab, cetuximab, rituximab, trastuzumab, alemtuzumab, ibritumomabtiuxetan, panitumumab, and combinations thereof.

In particular embodiments, the method includes simultaneously orsequentially administering to the subject a therapeutically effectiveamount of a cancer therapy selected from the group consisting of: (i)chemotherapy; (ii) photodynamic therapy; (iii) proton therapy; (iv)radiotherapy; (v) surgery; and combinations thereof.

In particular embodiments, the first immunotherapy, and the secondimmunotherapy if administered, is/are administered to the subject in theabsence of a cancer therapy selected from the group consisting of: (i)chemotherapy; (ii) photodynamic therapy; (iii) proton therapy; (iv)radiotherapy; (v) surgery; and combinations thereof.

In particular embodiments, the cancer is: (i) a cancer of the centralnervous system; (ii) a cancer that is associated with transplant and/orimmunosuppression; (iii) a cancer that is refractory to chemotherapy;(iv) a cancer that is refractory to photodynamic therapy; (v) a cancerthat is refractory to proton therapy; (vi) a cancer that is refractoryto radiotherapy; and (vii) a cancer that is refractory to surgery.

In particular embodiments, the cancer is a newly diagnosed, recurrent,and/or refractory cancer selected from the group consisting ofcelnasopharyngeal cancer, synovial cancer, hepatocellular cancer, renalcancer, cancer of connective tissues, melanoma, lung cancer, bowelcancer, colon cancer, rectal cancer, colorectal cancer, brain cancer,throat cancer, oral cancer, liver cancer, bone cancer, pancreaticcancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma,T-cell leukemia/lymphoma, neuroma, von Hippel-Lindau disease,Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile ductcancer, bladder cancer, ureter cancer, brain cancer, oligodendroglioma,neuroblastoma, meningioma, spinal cord tumor, bone cancer,osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknownprimary site, carcinoid, carcinoid of gastrointestinal tract,fibrosarcoma, breast cancer, Paget's disease, cervical cancer,colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer,head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, livercancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicularcancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skincancer, mesothelioma, multiple myeloma, ovarian cancer, endocrinepancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer,penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma,small intestine cancer, stomach cancer, thymus cancer, thyroid cancer,trophoblastic cancer, hydatidiform mole, uterine cancer, endometrialcancer, vagina cancer, vulva cancer, acoustic neuroma, mycosisfungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer,heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer,palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer,pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

In some aspects, the presently disclosed subject matter provides amethod of preventing or reducing the incidence of a relapse in a cancersubject in remission, the method comprising administering to the subjecta therapeutically effective amount of a metabolic reprogramming agent,wherein the metabolic reprogramming agent is selected from the groupconsisting of acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), azaserine,and 6-diazo-5-oxo-norleucine (DON), and 5-diazo-4-oxo-L-norvaline(L-DONV), and prodrugs thereof. In particular embodiments, at least onemetabolic reprogramming agent is a compound having any one of formula(I), formula (IIA), formula (IIB), or formula (III), below.

In particular embodiments, the metabolic reprogramming agent is: (i)administered to the subject post transplant; (ii) administered to thesubject post chemotherapy; (iii) administered to the subject postimmunotherapy; (iv) administered to the subject post photodynamictherapy; (v) administered to the subject post proton therapy; (vi)administered to the subject post radiotherapy; (vii) administered to thesubject post surgery; and combinations thereof.

Applicant has found that compounds of the disclosure having formula (I),formula (IIA), formula (IIB), and formula (III) are stable in plasma,liver microsomes, liver tissue, and gastrointestinal tissue, yet thesecompounds are cleaved in tumor cells to liberate DON in tumor tissue.The unexpected tumor-targeted properties of compounds having formula(I), formula (IIA), formula (IIB), and formula (III) result in asurprising improvement in therapeutic index for treating cancer with DONand provide the maximum therapeutic benefit to a subject in need of suchtreatment.

Applicant has also found unexpectedly that compounds of the disclosurehaving formula (I), formula (IIA), formula (IIB), and formula (III)exhibit unexpected enhanced CSF to plasma partitioning afteradministration, making them uniquely useful for the treatment of CNScancers such as glioblastoma, oligodendroglioma, neuroblastoma,meningioma, spinal cord tumor and metastatic cancer that has spread tothe central nervous system (CNS).

Applicant has also found unexpectedly that compounds of the disclosurehaving formula (I), formula (IIA), formula (IIB), and formula (III)condition tumors to be eliminated by checkpoint inhibitor therapy, e.g.with anti PD-1 antibodies, in subjects with cancer.

Applicant has also found unexpectedly that compounds of the disclosurehaving formula (I), formula (IIA), formula (IIB), and formula (III)enhance the response to checkpoint inhibitor therapy, e.g., with antiPD-1 antibodies.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Drawings as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1 is a line graph showing that metabolic reprogramming therapy withat least one metabolic reprogramming agent (e.g., DON) markedly inhibitslymphoma growth in a EL4 mouse lymphoma model, suggesting that bonemarrow derived tumors may be exquisitely susceptible to metabolicreprogramming therapy with at least one metabolic reprogramming agent(e.g., DON);

FIG. 2 is a bar graph showing that metabolic reprogramming therapy withat least one metabolic reprogramming agent (e.g., DON) has a modesteffect on inhibiting melanoma growth, which is a not a bone marrowderived tumor;

FIG. 3 is a graph showing that metabolic reprogramming therapy with atleast one metabolic reprogramming agent (e.g., DON) conditions B16melanoma to be killed by immunotherapy by inhibiting tumor infiltratingRegulatory T cells (Foxp3⁺);

FIG. 4 is an illustration showing the structures of DON and DON-basedprodrugs;

FIG. 5A is a bar graph and FIG. 5B is a line graph showing that DON (1)inhibits glutamine metabolism and GBM tumor growth in vivo. FIG. 5Ashows compound 1 (0.8 mg/kg, i.p.) inhibited glutamine metabolism asevidenced by increased endogenous glutamine concentrations in flank GBMtumors 2 hours post-administration relative to vehicle-treated controls;*p<0.05. FIG. 5B shows in efficacy studies, compared to Day 0 baseline,vehicle-treated mice exhibited significant growth of flank GBM tumorsduring the course of the experiment. By contrast, systemicadministration of 1 (0.8 mg/kg, i.p, q.d. days 1-6) caused a dramaticreduction in tumor size; ***p<0.001, ****p<0.0001. Note the bold numbersfollowing the terms “DON”, “DON prodrugs”, “DON-based prodrugs”, and thelike, refer to particular compounds disclosed in Table 1 below.

FIGS. 6A and 6B are lines graphs and FIG. 6C is a table showing in vivobrain and plasma pharmacokinetics of compound DON (1) following oraladministration of DON (1) and 5c in mice. 1 and 5c were dosed in mice at0.8 mg/kg equivalent, via oral gavage and plasma and brainconcentrations of compound 1 were evaluated via LC/MS. Oraladministration of compound 1 and 5c exhibited similar plasma and brainpharmacokinetic profiles due to complete and rapid metabolism of 5c to 1in mouse plasma;

FIG. 7A is a line graph, FIG. 7B is a bar graph, FIG. 7C is a table, anFIG. 7D is an illustration showing in vivo pharmacokinetics of DONfollowing i.v. administration of DON (1) and 5c in monkey plasma andCSF. 1 and 5c were dosed in two pigtail macaques at 1.6 mg/kg equivalentof 1 via i.v. administration and plasma (0.25-6 h) and CSF (30 min)concentrations of DON were evaluated via LC/MS. Relative to 1, 5cdelivered substantially lower DON plasma concentration. Unexpectedly,the reverse was observed in CSF, where 5c delivered significantly higherDON CSF concentrations, achieving a 10-fold enhanced CSF to plasma ratioat 30 minute post dose;

FIG. 8A is a Kaplan-Meier graph, and FIG. 8B, FIG. 8C, FIG. 8D, and FIG.8E are line graphs showing that 25 (5 day dosing starting on day 7) issuperior to CB-839 (30 day dosing starting day 1) in CT26 tumor model;

FIG. 9 is a line graph showing that 25 (4 days starting on day 6) issuperior to CB-839 (continuous twice daily dosing starting on day 1prior to engraftment) in a CT26 tumor model. FIG. 9 shows mice receiveddaily 25 (1.9 mg/kg) on days 6-9 vs BID glutaminase inhibitor on days1-15;

FIG. 10 is a line graph showing that 25 (daily days 7-22) is superior toCB-839 (continuous twice daily dosing days 1-29) in a 4T1 breast cancermodel. Mice received daily 25 (1.0 mg·kg/d) for days 7-22 as compared toBID glutaminase inhibitor for days 1-29;

FIGS. 11A-D and FIG. 11F are line graphs, and FIG. 11E is a Kaplan-Meiergraph showing that 25 dosing of 1 mg/kg following by 0.3 mg/kg leads toa complete and durable response in the MC38 tumor;

FIG. 12A, FIG. 12B, and FIG. 12D are line graphs, and FIG. 12C is aKaplan-Meier graph showing that 25 gives a robust response and improvedoverall survival in multiple tumor models including, for example, CT26Colon Cancer;

FIG. 13A, FIG. 13B, and FIG. 13D are line graphs, and FIG. 13C is a bargraph showing that 25 provides a robust response and improved overallsurvival in multiple tumor models including, for example, 4T1 breastcancer;

FIGS. 14A-G are line graphs showing that mice cured with 25 aloneimmunologically reject tumors upon re-challenge, demonstrating that 25monotherapy is immunotherapy;

FIG. 15A and FIG. 15C are line graphs and FIG. 15B and FIG. 15D areKaplan-Meier graphs showing that 25 monotherapy is immunotherapy;

FIGS. 16A-F are line graphs showing that glutamine inhibition (e.g.,DON) reduces the oxygen consumption and lactate production of tumorcells;

FIGS. 17A-F are graphs showing that glutamine inhibition (e.g., DON)improved the CD8/Treg ratio in the tumor and reduces hypoxia in theTILs;

FIGS. 18A-D are line graphs and FIG. 18E is a Kaplan-Meier graph showingthat 25 conditions the tumor to be eliminated by anti-PD1 therapy in theMC38 Model, and in particular that 25 unexpectedly rescues anti-PD1failures;

FIGS. 19A-C are line graphs and FIG. 19D is a Kaplan-Meier graph showingthat even in the more difficult CT26 model, 25 enhances the response toanti-PD1 therapy;

FIG. 20A is a line graph, and FIG. 20B is a Kaplan-Meier graph showingthat inhibiting glutamine metabolism also unexpectedly potentiates theanti-tumor response to adenosine A2a receptor (A2aR) blockade;

FIG. 21A is a line graph, FIG. 21B is an illustration, and FIG. 21C is aKaplan-Meier graph showing that inhibiting glutamine metabolismunexpectedly enhances the efficacy of adoptive cellular therapy (ACT) ina B16-OVA model;

FIG. 22A is a line graph, FIG. 22B is a bar graph, and FIG. 22C is atable showing the in vivo pharmacokinetics of DON following i.v.administration of DON (1) and 14b in monkey plasma and cerebrospinalfluid (CSF). 1 and 14b were dosed in two pigtail macaques at 1.6 mg/kgequivalent of 1 via i.v. administration and plasma (0.25-6 h) and CSF(30 min) concentrations of DON were evaluated via LC/MS. Relative to 1,14b delivered substantially lower DON plasma concentration. The reversewas observed in CSF, where 14b delivered significantly higher DON CSFconcentrations, achieving an unexpected 10-fold enhanced CSF to plasmaratio at 30 minute post dose;

FIG. 23 is a bar graph showing species specific plasma stability of(14b); 14b is stable in plasma of human, pig, dog and monkeys, butrapidly metabolized in mice;

FIG. 24 is an illustration showing exemplary structures of DON andDON-based prodrugs 25, 9, 38, 38a, 60, and 60a; different N-amino acidpromoeities (e.g, leucine, tryptophan) provide differential plasmas andmicrosomal stability;

FIG. 25A, FIG. 25B, FIG. 25C, and FIG. 25D are bar graphs showing the invitro plasma stability of DON prodrugs 25, 9, 38a and 60a. Metabolismoccurs via removal of N-protecting group; both ethyl and isopropylesters are stable in plasma of pigs and humans;

FIG. 26A, FIG. 26B, FIG. 26C, and FIG. 26D are bar graphs showing the invitro liver microsomal stability of DON prodrugs 25, 9, 38a and 60a; allprodrugs showed moderate-high stability in human and pig microsomes;

FIG. 27A, FIG. 27B, FIG. 27C, FIG. 27D, FIG. 27E, FIG. 27F, FIG. 27G,FIG. 27H, FIG. 27I, and FIG. 27J are bar graphs showing the results ofex-vivo studies in whole human and pig blood of 25, 9, 38a and 60a; DONprodrugs selectively deliver DON to PBMCs in both humans and pigs vsplasma; compared to DON, the PBMC/plasma ratio was unexpectedly enhanced10-100+ fold;

FIG. 28A FIG. 28B, FIG. 28C, FIG. 28D and FIG. 28E are line graphsshowing the results of pig in vivo studies with DON prodrugs of 25, 9,38a and 60a; DON prodrugs selectively deliver DON to PBMCs vs plasma;compared to DON, the PBMC/plasma ratio was expectedly enhanced 6- to10-fold;

FIG. 29A, FIG. 29B, and FIG. 29C are bar graphs showing the plasmastability of compound Methyl-POM 14b and its derivatives;

FIG. 30 is an illustration showing exemplary structures ofN-acylalkyloxy DON-based prodrug analogs for intracellular targeting andbrain penetration; the addition of steric bulk to the “bridge” mightresult in a slower hydrolysis;

FIG. 31 is a line graph showing that anti-PD1 monotherapy does not workin a 4T1 tumor model. 4T1 tumor cells (0.1 million) were injected intothe mammary fat pads of 8-week-old female BALB/c mice. Anti-PD1 (5mg/Kg) was administered on day 3, 5, 8, and 11 and tumor volume wasmeasured 2-3 times weekly until tumors were evaluated for tumorinfiltrating cells. Group: a-PD1 (d3, 5, 8, 11 100 ug/mouse)=5 mg/kgI.P.;

FIG. 32A is an illustration of mice and FIG. 32B is a line graphillustrating that compound 25 inhibits tumor growth. FIG. 32A shows miceafter 30 days inoculation (8 days drug free). FIG. 32B shows tumor areavs. days post injection with 4T1 tumor cells. Mice were treated withcompound 25 1 mg/kg every day (from d5-d22) or vehicle. 4T1 tumor cells(0.1 million) were injected into the mammary fat pads of 8-week-oldfemale BALB/c mice. Mice received vehicle (PBS) or 1 mg/kg compound 25daily from day 5 to day 22. Photos were taken on day 30 after tumorinoculation (FIG. 57A). Tumor volume was measured 2-3 times weekly untilWT mice were sacrificed (when size reached to 20 mm length or necrosisoccurred). Day 0: 100K 4T1 cells s.c. in 4th mammary pad. Day 5-22:Daily compound 25. The results show that compound 25 inhibited thegrowth of mammary carcinoma tumor cells.

FIG. 33A is an illustration of mice and FIG. 33B is a bar graphillustrating that compound 25 inhibits tumor growth. 4T1-Luc tumor cells(0.1 million) were injected into the mammary fat pads of 8-week-oldfemale BALB/c mice. Mice received vehicle (PBS) or 1 mg/kg compound 25daily from day 7. Anti-PD1 antibody (5 mg/Kg) was administered on day 5,8, and 12. Mice carrying 4T1-luc tumors are injected with Luciferin tomeasure luminescence from the tumor. IVIS imaging were taken on day 13.The results unexpectedly show that treatment with compound 25 as asingle agent inhibited mammary carcinoma cell proliferation better thananti-PD1.

FIG. 34A is a line graph and FIG. 34B is a bar graph showing thatcompound 25 inhibits tumor growth. FIG. 34B shows tumor weight (mg) onharvest day 21. 4T1-Luc tumor cells (0.1 million) were injected into themammary fat pads of 8-week-old female BALB/c mice. Mice received vehicle(PBS) or 1 mg/kg 25 daily from day 7 to day 16. Anti-PD1 antibody (5mg/Kg) was administered on day 5, 8, 12 and 17. Tumor volume wasmeasured 2-3 times weekly until tumors were evaluated for tumorinfiltrating cells on day 21. Tumor weights were measured on day 21. Onday 21, the PD1 group tumor size looks like it was reduced, however, theresult was not due to a tumor size reduction, but rather because one ofthe large tumor mice died;

FIGS. 35A-C are line graphs showing reduced CD11b+ cells and G-MDSCs in25 treated mice, demonstrating that metabolic reprogramming agents thatdecrease glutamine metabolic activity inhibit myeloid derived suppressorcells. 4T1-Luc tumor cells (0.1 million) were injected into the mammaryfat pads of 8-week-old female BALB/c mice. Mice received vehicle (PBS)or 1 mg/kg 25 daily from day 7 to day 16. Anti-PD1 (5 mg/Kg) wasadministered on day 5, 8, 12 and 17. Percentages of myeloid-derivedsuppressor cell (MDSC) from circulating blood were monitored on day 6, 9and 17 by flow cytometry with CD11b and Ly6C/Ly6G. Granulocytic MDSC(G-MDSC): CD11b+Ly6g+ Lytic lo Monocytic MDSC (Mo-MDSC): CD11b+Ly6G-Ly6cHi;

FIG. 36 is a graph showing increased TNF alpha in 25 treated mice,demonstrating that metabolic reprogramming agents that decreaseglutamine metabolic activity increase inflammatory tumor associatedmacrophages (TAM). 4T1-Luc tumor cells (0.2 million) were injected intothe mammary fat pads of 8-week-old female BALB/c mice. Mice receivedvehicle (PBS) or 1 mg/kg 25 daily from day 7 to day 16. Anti-PD1 (5mg/Kg) was administered on day 5, 8, 12 and 17. Tumors were evaluatedfor tumor infiltrating cells on day 21. Cells were seeded on plates andgolgi plug 200 ul were added to inhibit cytokine secretion for overnight (no additional stimulation). Tumor associated macrophages markers:Live/CD45+/CD11b+/F4-80+/CD8− for flow cytometry analysis. Macrophagesderived from tumor. The cells were incubated with Golgi plug o.n. w/ostim. TAM: Live/CD45+/CD11b+/F4-80+/CD8-;

FIG. 37A is an illustration, FIG. 37B is a graph, and FIG. 37C is anillustration showing reduced fibrocytes in 25 treated mice,demonstrating that metabolic reprogramming agents that decreaseglutamine metabolic activity inhibit bone marrow derived fibrocyteswhich are thought to play a role in inhibiting immunotherapy, as well asgenerating an extracellular matrix that surrounds tumors and inhibitschemotherapy. 4T1-Luc tumor cells (0.1 million) were injected into themammary fat pads of 8-week-old female BALB/c mice. Mice received vehicle(PBS) or 1 mg/kg 25 daily from day 7 to day 16. Anti-PD1 (5 mg/Kg) wasadministered on day 5, 8, 12 and 17. Tumors were evaluated for tumorinfiltrating cells on day 21. Fibrocytes markers:CollagenI+CD11b+CD45+live were used for flow cytometry analysis.

FIG. 38A is a line graph demonstrating different DON plasma profiles inMonkey for DON and compound 14b.

FIG. 38B is a bar graph showing that compound 14b exhibited enhancedCSF:plasma ratio of DON in Monkey.

FIG. 39A is a line graph demonstrating different DON plasma profiles inswine for DON, compound 14b and compound 47.

FIG. 39B is a bar graph showing that compounds 14b and 47 exhibitedenhanced CSF delivery of DON at 60 min post-administration in swine

FIG. 39C is a bar graph showing that compounds 14b and 47 exhibitedenhanced CSF:plasma ratio of DON at 60 min post-administration in swine.

FIG. 40A is a line graph showing significant inhibition of tumor growthwith compound 25 in EO771 tumor-bearing mice.

FIG. 40B is a line graph showing an increase in survival time withcompound 25 in EO771 tumor-bearing mice.

FIG. 40C is a line graph showing EO771 tumor growth in mice treated withvehicle.

FIG. 40D is a line graph showing EO771 tumor growth in mice treated withanti-PD1.

FIG. 40E is a line graph showing EO771 tumor growth in mice treated withcompound 25.

FIG. 40F is a line graph showing EO771 tumor growth in mice treated withcombination of compound 25 and anti-PD-1.

FIG. 40G is a line graph showing an increase in survival time with thecombination of compound 25 and anti-PD-1 in EO771 tumor-bearing mice.

FIG. 41A is a line graph showing 4T1 tumor cells tumors were resistantto treatment with anti-PD1, anti-CTLA4, or combination of anti-PD1 andanti-CTLA4.

FIG. 41B is a graph showing percentages of Mo-MDSC (monocytic MDSC:CD11b+F4/80-Ly6ChiLy6Gneg) live cells from the blood.

FIG. 41C is a graph showing percentages of PMN-MDSC+TAN(CD11b+F4/80-Ly6CloLy6Ghi) live cells from the blood.

FIG. 41D is a graph showing 4T1 tumor weight in mice treated withvehicle, anti-PD1, anti-CTLA4, and combination of anti-PD1 andanti-CTLA4.

FIG. 41E is a graph showing the ratio of CD8 to MDSC in mice treatedwith vehicle, anti-PD1, anti-CTLA4, and combination of anti-PD1 andanti-CTLA4.

FIG. 41F is a graph showing the percentages of PMN-MDSC+TAN(CD11b+F4/80-Ly6CloLy6Ghi) in mice treated with vehicle, anti-PD1,anti-CTLA4, and combination of anti-PD1 and anti-CTLA4.

FIG. 41G is a graph showing percentages of Mo-MDSC (monocytic MDSC:CD11b+F4/80-Ly6ChiLy6Gneg) in mice treated with vehicle, anti-PD1,anti-CTLA4, and combination of anti-PD1 and anti-CTLA4.

FIG. 41H is a line graph showing 4T1 tumor volume in mice treated withvehicle.

FIG. 41I is a line graph showing 4T1 tumor volume in mice treated withanti-CTLA4 and anti-PD1.

FIG. 41J is a line graph showing that the compound 25 treated group inthe 4T1 tumor model displayed slow tumor growth and increased survival.

FIG. 41K is a line graph showing that combination of compound 25,anti-PD1 and anti-CTLA4 in the 4T1 tumor model further slowed tumorgrowth compared to the compound 25 alone group.

FIG. 41L is a line graph showing an increase in survival time with thecombination of compound 25, anti-PD1 and anti-CTLA4 in the 4T1 tumormodel.

FIG. 42A is an illustration showing spontaneous lung metastasis in 4T1tumor-bearing mice analyzed by inflation with 15% india ink to quantifytumor nodules.

FIG. 42B is a graph showing numbers of spontaneous 4T1 lung metastesesin non-treated (NT) mice and mice treated with compound 25.

FIG. 43A is a line graph showing tumor growth curves of MC38-bearingmice treated with vehicle and with compound 25.

FIG. 43B is a line graph showing tumor volume of MC38-bearing micetreated with vehicle and with compound 25.

FIG. 43C is a line graph showing survival curves of MC38-bearing micetreated with vehicle and with compound 25.

FIG. 44A is a line graph showing tumor growth curves of CT26tumor-bearing mice treated with vehicle and with compound 25.

FIG. 44B is a line graph showing survival curves of CT26 tumor-bearingmice treated with vehicle and with compound 25.

FIG. 45A is a line graph showing tumor growth curves of B16tumor-bearing mice treated with vehicle and with compound 25.

FIG. 45B is a line graph showing survival curves of B16 tumor-bearingmice treated with vehicle and with compound 25.

FIG. 46A is a line graph showing tumor growth curves of EL4tumor-bearing mice treated with vehicle and with compound 25.

FIG. 46B is a line graph showing survival curves of EL4 tumor-bearingmice treated with vehicle and with compound 25.

FIG. 47A is a line graph showing tumor growth of MC38 bearing C57BL/6mice treated with vehicle, anti-PD-1, compound 25, or combination ofcompound 25 and anti-PD-1, beginning on day 10 after tumor inoculation.

FIG. 47B is a line graph showing survival of MC38 bearing C57BL/6 micetreated with vehicle, anti-PD-1, compound 25, or combination of compound25 and anti-PD-1, beginning on day 10 after tumor inoculation.

FIG. 47C is a line graph showing tumor cell growth of MC38 bearingC57BL/6 mice treated with vehicle.

FIG. 47D is a line graph showing tumor cell growth of MC38 bearingC57BL/6 mice treated with anti-PD-1.

FIG. 47E is a line graph showing tumor cell growth of MC38 bearingC57BL/6 mice treated with compound 25.

FIG. 47F is a line graph showing tumor cell growth of MC38 bearingC57BL/6 mice treated with combination of compound 25 and anti-PD-1beginning on day 10 after tumor inoculation.

FIG. 48A is a line graph showing tumor growth of CT26 bearing BALB/cmice treated with vehicle, anti-PD-1, compound 25, or combination ofcompound 25 and anti-PD-1, beginning on day 10 after tumor inoculation.

FIG. 48B is a line graph showing survival of CT26 bearing BALB/c micetreated with vehicle, anti-PD-1, compound 25, or combination of compound25 and anti-PD-1, beginning on day 10 after tumor inoculation.

FIG. 48C is a line graph showing tumor growth of CT26 bearing BALB/cmice treated with vehicle.

FIG. 48D is a line graph showing tumor growth of CT26 bearing BALB/cmice treated with anti-PD-1.

FIG. 48E is a line graph showing tumor growth of CT26 bearing BALB/cmice treated with compound 25.

FIG. 48F is a line graph showing tumor growth of CT26 bearing BALB/cmice treated with combination of compound 25 and anti-PD-1, beginning onday 10 after tumor inoculation.

FIG. 49A is a line graph showing tumor growth of MC38 rechallenged mice.

FIG. 49B is a line graph showing tumor growth of MC38-bearing C57BL/6wild type.

FIG. 49C is a line graph showing tumor growth of MC38-bearing C57BL/6RAG^(−/−) mice treated with compound 25 for 14 days.

FIG. 49D is a line graph showing survival of MC38-bearing C57BL/6 wildtype and RAG−/− mice treated with compound 25 for 14 days.

FIG. 50A is a line graph showing tumor growth of B16OVA-bearing C57BL/6mice treated with compound 25 (1 mg/kg) or vehicle on days 7-9 aftertumor inoculation. The mice received 1.5×10⁶ activated OT1 T cells onday 10.

FIG. 50B is a line graph showing survival of B16OVA-bearing C57BL/6 micetreated with compound 25 (1 mg/kg) or vehicle on days 7-9 after tumorinoculation. The mice received 1.5×10⁶ activated OT1 T cells on day 10.

FIG. 50C is a line graph showing tumor growth of B16OVA-bearing C57BL/6mice treated with vehicle on days 7-9 after tumor inoculation. The micereceived 1.5×10⁶ activated OT1 T cells on day 10.

FIG. 50D is a line graph showing tumor growth of B16OVA-bearing C57BL/6mice treated with compound 25 (1 mg/kg) on days 7-9 after tumorinoculation. The mice received 1.5×10⁶ activated OT1 T cells on day 10.

FIG. 51A is a line graph showing the tumor size of 3LL tumor-bearingmice treated with compound 25.

FIG. 51B is a bar graph showing the live cell percentages of Ly6c loLy6g hi, Ly6c hi Grl, CD8+, and CD4+ of cells from blood in 3LL tumorbearing mice with no treatment (NT) and with compound 25.

FIG. 51C is a graph showing the ratio of CD8 cells to MDSCs+ and TANs inblood in 3LL tumor bearing mice with no treatment (NT) and with compound25.

FIG. 51D is two graphs showing the ratio of CD8 cells to MDSCs+ and TANsin TIL in 3LL tumor bearing mice with no treatment (NT) and withcompound 25.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the presently disclosed subject matter areshown. Like numbers refer to like elements throughout. The presentlydisclosed subject matter may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. Indeed, many modifications andother embodiments of the presently disclosed subject matter set forthherein will come to mind to one skilled in the art to which thepresently disclosed subject matter pertains having the benefit of theteachings presented in the foregoing descriptions and the associatedFigures. Therefore, it is to be understood that the presently disclosedsubject matter is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims.

The presently disclosed subject matter demonstrates that certainconditions, diseases, and/or disorders involve metabolicallyreprogrammed cells whose activation, function, growth, proliferation,and/or survival in an abnormal, harmful, and/or unhealthy state dependon increased activity of at least one, at least two, or at least threemetabolic pathways selected from the group consisting of glutaminemetabolism, glycolysis, and fatty acid synthesis. It should beappreciated that the abnormal, harmful, and/or unhealthy state of thecell refers to its effect on or relative to the subject whose cells areaffected by the condition, disease, or disorder rather than on orrelative to the cell itself which exhibits an increased ability tothrive in the abnormal, harmful, and/or unhealthy state in a manner thatis believed to be proportionate to the increase in the activity of atleast one, at least two, or at least three metabolic pathways (e.g.,glutamine metabolism, glycolysis, and/or fatty acid synthesis).

The presently disclosed subject matter have demonstrated that certain ofsuch conditions, diseases, and/or disorders, referred to herein as“metabolic reprogramming disorders,” are amenable to treatment using atleast one, at least two, or at least three metabolic reprogrammingagents that decrease activity of at least one, at least two, or at leastthree metabolic pathways selected from the group consisting of glutaminemetabolism, glycolysis, and fatty acid synthesis. In some instances, themetabolic reprogramming disorders comprise conditions, diseases, ordisorders that involve aberrant and/or excessive glutamine metabolism,aberrant and/or excessive glycolysis, or aberrant and/or excessive fattyacid synthesis.

As used herein, the term “excessive glutamine metabolism” means anincrease in the amount of glutamine metabolic activity in a subject witha condition, disease, or disorder (e.g., a metabolic reprogrammingdisorder) as compared to the amount of glutamine metabolic activity in asubject without a similar disease or condition, such as an increase ofapproximately 100%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000%, or more. As used herein, the term “aberrant glutaminemetabolism” means a change in the biological activity of glutamine in asubject with a condition, disease or disorder (e.g., a metabolicreprogramming disorder) as compared to the glutamine activity in asubject without a similar condition, disease, or disorder, such asincreased utilization of glutamine in the growth and/or proliferation ofmalignant, neoplastic, or other pathologic cellular processes (e.g.,immune disorders, neurodegenerative disorders, inflammatory disorders,etc.).

As used herein, the term “excessive glycolysis metabolism” means anincrease in the amount of glycolytic metabolic activity in a subjectwith a condition, disease, or disorder (e.g., a metabolic reprogrammingdisorder) as compared to the amount of glycolytic metabolic activity ina subject without a similar disease or condition, such as an increase ofapproximately 100%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000%, or more. As used herein, the term “aberrant glycolyticmetabolism” means a change in the biological activity of glycolysis in asubject with a condition, disease or disorder (e.g., a metabolicreprogramming disorder) as compared to the glycolytic activity in asubject without a similar condition, disease, or disorder, such asincreased utilization of glucose in the growth and/or proliferation ofmalignant, neoplastic, or other pathologic cellular processes (e.g.,immune disorders, neurodegenerative disorders, inflammatory disorders,etc.).

As used herein, the term “excessive fatty acid synthesis” means anincrease in the amount of fatty acid synthesis in a subject with acondition, disease, or disorder (e.g., a metabolic reprogrammingdisorder) as compared to the amount of fatty acid synthesis in a subjectwithout a similar condition, disease, or disorder, such as an increaseof approximately 100%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000%, or more. As used herein, the term “aberrant fatty acidsynthesis” means a change in the biological activity of fatty acidsynthesis in a subject with a condition, disease or disorder (e.g., ametabolic reprogramming disorder) as compared to the fatty acidsynthesis in a subject without a similar condition, disease, ordisorder, such as increased utilization of fatty acids in the growthand/or proliferation of malignant, neoplastic, or other pathologiccellular processes (e.g., immune disorders, neurodegenerative disorders,inflammatory disorders, etc.).

As used herein, a “metabolically reprogrammed” cell refers to a cell inwhich the activity of at least one, at least two, or at least threemetabolic pathways (e.g., glutamine metabolism, glycolysis, and fattyacid synthesis) has increased in response to the cells energetic andbiosynthetic demands placed on the cell in order for the cell to becomeactivated, function, grow, proliferate, and/or survive in the abnormal,harmful, and/or unhealthy state. As used herein, a “metabolicreprogramming agent” refers to an agent that is capable of reversing themetabolic reprogramming of a cell from a cell whose activation,function, growth, proliferation, and/or survival in an abnormal,harmful, and/or unhealthy state depends on increased activity of atleast one, at least two, or at least three metabolic pathways (e.g.,glutamine metabolism, glycolysis, and fatty acid synthesis) to a cellthat has a decreased capacity or has lost its ability to thrive (e.g.,activate, function, grow, proliferate, and/or survive) in the abnormal,harmful and/or unhealthy state. In some contexts, a “metabolicreprogramming agent” inhibits at least one of, at least two of, or allof aberrant and/or excessive glutamine metabolism, aberrant and/orexcessive glycolysis, and aberrant and/or excessive fatty acidsynthesis.

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the inventions are shown. Like numbers referto like elements throughout. The presently disclosed subject matter maybe embodied in many different forms and should not be construed aslimited to the embodiments set forth herein; rather, these embodimentsare provided so that this disclosure will satisfy applicable legalrequirements. Indeed, many modifications and other embodiments of thepresently disclosed subject matter set forth herein will come to mind toone skilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions and the associated Figures. Therefore, it is to beunderstood that the presently disclosed subject matter is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims.

I. Methods of Treatment Using Metabolic Reprogramming Agents

In an aspect, the presently disclosed subject matter provides a methodfor treating a subject having a condition, disease, or disorder thatinvolves metabolically reprogrammed cells whose activation, function,growth, proliferation, and/or survival depends on increased activity ofat least one metabolic pathway selected from the group consisting ofglutamine metabolism, glycolysis, and fatty acid synthesis, the methodcomprising administering to the subject at least one metabolicreprogramming agent that decreases activity of at least one metabolicpathway selected from the group consisting of glutamine metabolism,glycolysis, and fatty acid synthesis in an amount effective to treat thecondition, disease, or disorder.

In some aspects, the presently disclosed subject matter provides amethod for treating a subject having a condition, disease, or disorderthat involves at least one of aberrant and/or excessive glutaminemetabolism, aberrant and/or excessive glycolysis, or aberrant and/orexcessive fatty acid synthesis, the method comprising administering tothe subject at least one metabolic reprogramming agent that decreasesactivity of at least one metabolic pathway selected from the groupconsisting of glutamine metabolism, glycolysis, and fatty acid synthesisin an amount effective to treat the condition, disease, or disorder.

In general, the presently disclosed methods result in a decrease in theseverity of a condition, disease, or disorder (e.g., a metabolicreprogramming disorder) in a subject. The term “decrease” is meant toinhibit, suppress, attenuate, diminish, arrest, or stabilize a symptomof the condition, disease, or disorder. As used herein, the terms“treat,” “treating,” “treatment,” and the like refer to reducing orameliorating a disease or condition, and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disease or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

In some embodiments, the method comprises administering to the subjectat least two metabolic reprogramming agents that decrease the activityof at least two metabolic pathways selected from the group consisting ofglutamine metabolism, glycolysis, and fatty acid synthesis in an amounteffective to treat the condition, disease, or disorder. In otherembodiments, the method comprises administering to the subject at leastthree metabolic reprogramming agents that each decrease the activity ofa different metabolic pathway selected from the group consisting ofglutamine metabolism, glycolysis, and fatty acid synthesis in an amounteffective to treat the condition, disease, or disorder.

The terms “subject” and “patient” are used interchangeably herein. Thesubject treated by the presently disclosed methods, uses, metabolicreprogramming agents and compositions comprising those agents in theirmany embodiments is desirably a human subject, although it is to beunderstood that the methods described herein are effective with respectto all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject formedical purposes, such as for the treatment of an existing condition ordisease or the prophylactic treatment for preventing the onset of acondition or disease, or an animal subject for medical, veterinarypurposes, or developmental purposes. Suitable animal subjects includemammals including, but not limited to, primates, e.g., humans, monkeys,apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,e.g., sheep and the like; caprines, e.g., goats and the like; porcines,e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras,and the like; felines, including wild and domestic cats; canines,including dogs; lagomorphs, including rabbits, hares, and the like; androdents, including mice, rats, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a condition or disease.

A. Cancer

Aspects of the invention involve the use of at least one, at least two,or at least three metabolic reprogramming agents, alone, or optionallytogether in combination with a chemotherapeutic agent, animmunotherapeutic agent, and/or a radiotherapeutic agent, for thetreatment of a cancer. Accordingly, in some embodiments, the condition,disease, or disorder is a cancer. In such embodiments, the metabolicallyreprogrammed cells comprise malignant or cancerous cells. Examples ofmalignant or cancer cells whose activation, function, growth,proliferation, and/or survival in an abnormal, harmful, or unhealthystate depends on increased metabolic activity of at least one, at leasttwo, or at least three metabolic pathways selected from the groupconsisting of glutamine metabolism, glycolysis and fatty acid synthesisinclude, but are not limited to, cMyc-dependent cancer cells,glutamine-dependent cancer cells, and combinations thereof. As usedherein, a “glutamine-dependent cancer cell” is a cancer cell in whichglutamine is an important fuel source for cellular energy in the cancercell (e.g., hematopoietic tumors, hepatomas, Ehrilich carcinoma (seeHuber et al., “Uptake of glutamine antimetabolites6-diazo-5-oxo-L-norleucine (DON) and acivicin in sensitive and resistanttumor cell lines,” Int. J. Cancer. 1988; 41:752-755)). As used herein,cMyc-dependent cancer cells” refers to cancer cells exhibitingactivation, overexpression and/or amplification of c-Myc. In somecontexts, a “Myc-dependent cancer” is a cancer in which c-Myc plays arole in increased glutamine metabolism in the cancer cells, i.e.,cMyc-dependent glutamine addicted cancer cells. Examples ofMyc-dependent cancers include, without limitation, lymphoma,neuroblastoma, and small cell lung cancer.

Aspects of the presently disclosed subject matter further involve theuse of at least one metabolic reprogramming agent (e.g., a metabolicreprogramming agent that decreases glutamine metabolism) as a cancermaintenance thereapy. As used herein, “cancer maintenance therapy”refers to a therapy administered to a cancer patient who is in cancerremission.

Accordingly, in one aspect, the presently disclosed subject matterprovides a method of preventing a relapse or reducing the incidence ofrelapse of a cancer subject in remission, the method comprisingadministering to the subject a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism. Insome embodiments, at least one metabolic reprogramming agent is acompound having any one of formula (I), formula (IIA), formula (IIB), orformula (III), below. As used herein, “remission” includes partial andcomplete remission and refers to a decrease in or disappearance of signsand symptoms of cancer. “Partial remission” means that the cancerresponded to treatment with the primary therapy, but at least a portionof the tumor and/or at least a portion of the cancerous cells are stillpresent in the subject, for example, at least 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, or 49% of a measurable tumor and/or measurablecancerous cells are still present in the subject post-therapy. “Completeremission” means that the subject shows no signs or symptoms of cancer,for example, after a healthcare provider has used the most accurate andup-to-date tests available to detect the cancer and is unable to detectany signs or symptoms of cancer. It is to be understood that cancerouscells might still exist in a subject in complete remission at levelsthat are undetectable.

In some embodiments, the metabolic reprogramming agent is administeredto the subject post transplant. As used herein, “post transplant” refersto a subject that has recently received a cell, tissue, or organtransplantation, including for example, subjects receivingimmunosuppressive agents to prevent, or reduce the risk and/or severityof, a transplant rejection. In some embodiments, the metabolicreprogramming agent is administered to the subject post chemotherapy. Insome embodiments, the metabolic reprogramming agent is administered tothe subject post immunotherapy. In some embodiments, the metabolicreprogramming agent is administered to the subject post photodynamictherapy. In some embodiments, the metabolic reprogramming agent isadministered to the subject post proton therapy. In some embodiments,the metabolic reprogramming agent is administered to the subject postradiotherapy. In some embodiments, the metabolic reprogramming agent isadministered to the subject post surgery; and combinations thereof. Insome embodiments, the metabolic reprogramming agent is administered tothe subject at two or more of post transplant, post chemotherapy, postimmunotherapy, post photodynamic therapy, post proton therapy, postradiotherapy, post surgery, and combinations thereof.

As used herein, a “cancer” in a subject refers to the presence of cellspossessing characteristics typical of cancer-causing cells, for example,uncontrolled proliferation, loss of specialized functions, immortality,significant metastatic potential, significant increase in anti-apoptoticactivity, rapid growth and proliferation rate, and certaincharacteristic morphology and cellular markers. In some circumstances,cancer cells will be in the form of a tumor; such cells may existlocally within an animal, or circulate in the blood stream asindependent cells, for example, leukemic cells. A “tumor,” as usedherein, refers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all precancerous and cancerous cells andtissues. A “solid tumor”, as used herein, is an abnormal mass of tissuethat generally does not contain cysts or liquid areas. A solid tumor maybe in the brain, colon, breasts, prostate, liver, kidneys, lungs,esophagus, head and neck, ovaries, cervix, stomach, colon, rectum,bladder, uterus, testes, and pancreas, as non-limiting examples. In someembodiments, the solid tumor regresses or its growth is slowed orarrested after the solid tumor is treated with the presently disclosedmethods. In other embodiments, the solid tumor is malignant. In someembodiments, the cancer comprises Stage 0 cancer. In some embodiments,the cancer comprises Stage I cancer. In some embodiments, the cancercomprises Stage II cancer. In some embodiments, the cancer comprisesStage III cancer. In some embodiments, the cancer comprises Stage IVcancer. In some embodiments, the cancer is refractory and/or metastatic.For example, the cancer may be refractory to treatment withradiotherapy, chemotherapy or monotreatment with immunotherapy.

In particular embodiments, the cancer is a cancer of the central nervoussystem (CNS cancer). It is believed that certain of the presentlydisclosed metabolic reprogramming agents and compositions areparticularly useful in the treatment of CNS cancers and cancers of CNSorigin. In particular, the data described in FIG. 22A, FIG. 22B, FIG.22C, FIG. 29A, FIG. 29B, FIG. 29C and FIG. 30 unexpectedly demonstratethat certain metabolic reprogramming agents (e.g., prodrugs of glutamineanalogs, e.g., DON prodrugs, e.g., a compound having any one of formula(I), formula (IIA), formula (IIB), or formula (III), below.) effectivelytarget and deliver DON to the brain, for example, achieving as much as a10-fold enhanced CSF to plasma ratio at 30 minute post dosing.Accordingly, certain of the presently disclosed metabolic reprogrammingagents are contemplated for use as cancer therapy (e.g., maintenancetherapy), immunotherapy, and an enhancement to immunotherapy, for thetreatment of CNS cancers and cancers of CNS origin.

Exemplary CNS cancers treatable with the presently disclosed methods,compositions and agents include, without limitation, gliomas,astrocytomas, oligodendrogliomas, ependymoas, mixed gliomas (e.g.,oligoastrocytomas), meningiomas (e.g., atypical, invasive, anaplastic,etc.), medulloblastomas, gangliogliomas, schwannomas (neuroliemmomas),craniopharyngiomas, chordomas, non-Hodgkin lymphoma of CNS origin, and,pituitary tumors. In particular embodiments, the CNS cancer comprisesglioblastoma multiform (GBM).

In particular embodiments, the cancer is a cancer that is associatedwith transplant and/or immunosuppression. It is well known that organtransplants (e.g., kidney, liver, heart, lung etc.) in the United Statesare at high risk of developing various types of cancer (see, e.g.,Engels et al. 2011). In some instances, the cancer risk is elevated forinfection-related cancer due to immunosuppression, for example, becauseof medications administered to suppress the immune system and preventtransplant rejection (e.g., organ). In some embodiments, the cancerassociated with transplant and/or immunosuppression is related to aninfectious agent. Examples of cancers associated with transplant and/orimmunosuppression include, without limitation, anal cancer, Kaposisarcoma, kidney cancer, liver cancer, lung cancer, melanoma,non-Hodgkin's lymphoma, and thyroid cancer. In particular embodiments,the subject is a child or elderly adult transplant recipient (e.g.,liver, heart, kidney, etc.) who may or may not be infected withEpstein-Barr virus. In particular embodiments, the cancer is a cancerthat is refractory to chemotherapy. In particular embodiments, thecancer is a cancer that is refractory to photodynamic therapy. Inparticular embodiments, the cancer is a cancer that is refractory toproton therapy.

In particular embodiments, the cancer is a cancer that is refractory toradiotherapy.

In particular embodiments, the cancer is a cancer that is refractory tosurgery.

Cancer as used herein includes newly diagnosed or recurrent and/orrefractory cancers, including without limitation, acute lymphoblasticleukemia, acute myelogenous leukemia, advanced soft tissue sarcoma,brain cancer, metastatic or aggressive breast cancer, breast carcinoma,bronchogenic carcinoma, choriocarcinoma, chronic myelocytic leukemia,colon carcinoma, colorectal carcinoma, Ewing's sarcoma, gastrointestinaltract carcinoma, glioma, glioblastoma multiforme, head and neck squamouscell carcinoma, hepatocellular carcinoma, Hodgkin's disease,intracranial ependymoblastoma, large bowel cancer, leukemia, livercancer, lung carcinoma, Lewis lung carcinoma, lymphoma, malignantfibrous histiocytoma, a mammary tumor, melanoma, mesothelioma,neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, apontine tumor, premenopausal breast cancer, prostate cancer,rhabdomyosarcoma, reticulum cell sarcoma, sarcoma, small cell lungcancer, a solid tumor, stomach cancer, testicular cancer, and uterinecarcinoma.

In particular embodiments, the cancer treated is a newly diagnosed, orrecurrent, and/or refractory cancer selected from the group consistingof celnasopharyngeal cancer, synovial cancer, hepatocellular cancer,renal cancer, cancer of connective tissues, melanoma, lung cancer, bowelcancer, colon cancer, rectal cancer, colorectal cancer, brain cancer,throat cancer, oral cancer, liver cancer, bone cancer, pancreaticcancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma,T-cell leukemia/lymphoma, neuroma, von Hippel-Lindau disease,Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile ductcancer, bladder cancer, ureter cancer, brain cancer, oligodendroglioma,neuroblastoma, meningioma, spinal cord tumor, bone cancer,osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknownprimary site, carcinoid, carcinoid of gastrointestinal tract,fibrosarcoma, breast cancer, Paget's disease, cervical cancer,colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer,head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, livercancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicularcancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skincancer, mesothelioma, multiple myeloma, ovarian cancer, endocrinepancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer,penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma,small intestine cancer, stomach cancer, thymus cancer, thyroid cancer,trophoblastic cancer, hydatidiform mole, uterine cancer, endometrialcancer, vagina cancer, vulva cancer, acoustic neuroma, mycosisfungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer,heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer,palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer,pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

In some embodiments, the condition, disease, or disorder is lymphoma.Accordingly, in an aspect the presently disclosed subject matterprovides a method for the treatment of lymphoma in a subject in needthereof, the method comprising administering to the subject at least onemetabolic reprogramming agent that decreases glutamine metabolism in anamount effective to treat lymphoma in the subject.

In some embodiments, the condition, disease, or disorder is melanoma.Accordingly, in an aspect the presently disclosed subject matterprovides a method for the treatment of melanoma in a subject in needthereof, the method comprising administering to the subject at least onemetabolic reprogramming agent that decreases glutamine metabolism in anamount effective to treat melanoma in the subject.

In some embodiments, the methods include administering to the subject aneffective amount of radiotherapy. In some embodiments, the methodsinclude administering of the subject an effective amount ofimmunotherapy (e.g., a second immunotherapy). In some embodiments, themethods include administering to the subject an effective amount ofphotodynamic therapy. In some embodiments, the methods includeadministering to the subject an effective amount of proton therapy. Insome embodiments, the methods include surgically resecting at least aportion of a tumor before, during, or after treatment with the at leastone, at least two, or at least three metabolic reprogramming agents, andoptionally at least one chemotherapeutic agent, immunotherapeutic agent,and/or radiotherapeutic agent.

In some embodiments, the condition, disease, or disorder is a newlydiagnosed, or recurrent and/or refractory cancer selected from the groupconsisting of acute lymphoblastic leukemia, acute myelogenous leukemia,advanced soft tissue sarcoma, brain cancer, metastatic or aggressivebreast cancer, breast carcinoma, bronchogenic carcinoma,choriocarcinoma, chronic myelocytic leukemia, colon carcinoma,colorectal carcinoma, Ewing's sarcoma, gastrointestinal tract carcinoma,glioma, glioblastoma multiforme, head and neck squamous cell carcinoma,hepatocellular carcinoma, Hodgkin's disease, intracranialependymoblastoma, large bowel cancer, leukemia, liver cancer, lungcarcinoma, Lewis lung carcinoma, lymphoma, malignant fibroushistiocytoma, a mammary tumor, melanoma, mesothelioma, neuroblastoma,osteosarcoma, ovarian cancer, pancreatic cancer, a pontine tumor,premenopausal breast cancer, prostate cancer, rhabdomyosarcoma,reticulum cell sarcoma, sarcoma, small cell lung cancer, a solid tumor,stomach cancer, testicular cancer, and uterine carcinoma.

In some embodiments, the cancer is not acute lymphoblastic leukemia. Insome embodiments, the cancer is not acute myelogenous leukemia. In someembodiments, the cancer is not advanced soft tissue sarcoma. In someembodiments, the cancer is not brain cancer. In some embodiments, thecancer is not metastatic or aggressive breast cancer. In someembodiments, the cancer is not breast carcinoma. In some embodiments,the cancer is not bronchogenic carcinoma. In some embodiments, thecancer is not choriocarcinoma. In some embodiments, the cancer is notchronic myelocytic leukemia. In some embodiments, the cancer is notcolon carcinoma. In some embodiments, the cancer is not colorectalcarcinoma. In some embodiments, the cancer is not Ewing's sarcoma. Insome embodiments, the cancer is not gastrointestinal tract carcinoma. Insome embodiments, the cancer is not glioma. In some embodiments, thecancer is not glioblastoma multiforme. In some embodiments, the canceris not head and neck squamous cell carcinoma. In some embodiments, thecancer is not hepatocellular carcinoma. In some embodiments, the canceris not Hodgkin's disease. In some embodiments, the cancer is notintracranial ependymoblastoma. In some embodiments, the cancer is notlarge bowel cancer. In some embodiments, the cancer is not leukemia. Insome embodiments, the cancer is not liver cancer. In some embodiments,the cancer is not lung carcinoma. In some embodiments, the cancer is notLewis lung carcinoma. In some embodiments, the cancer is not lymphoma.In some embodiments, the cancer is not malignant fibrous histiocytoma.In some embodiments, the cancer is not a mammary tumor. In someembodiments, the cancer is not melanoma. In some embodiments, the canceris not mesothelioma. In some embodiments, the cancer is notneuroblastoma. In some embodiments, the cancer is not osteosarcoma. Insome embodiments, the cancer is not ovarian cancer. In some embodiments,the cancer is not pancreatic cancer. In some embodiments, the cancer isnot a pontine tumor. In some embodiments, the cancer is notpremenopausal breast cancer. In some embodiments, the cancer is notprostate cancer. In some embodiments, the cancer is notrhabdomyosarcoma. In some embodiments, the cancer is not reticulum cellsarcoma. In some embodiments, the cancer is not sarcoma. In someembodiments, the cancer is not small cell lung cancer. In someembodiments, the cancer is not a solid tumor. In some embodiments, thecancer is not stomach cancer. In some embodiments, the cancer is nottesticular cancer. In some embodiments, the cancer is not uterinecarcinoma.

B. Immunotherapy

Aspects of the presently disclosed subject matter involve the use of atleast one, at least two, or at least three metabolic reprogrammingagents, alone, or optionally together in combination with an additionalimmunotherapy (e.g., checkpoint blockade, adoptive cellular therapy(ACT), vaccines (e.g., tumor vaccines), passive immunotherapyantibodies, for the treatment of cancer.

Accordingly, in one aspect, the presently disclosed subject matterprovides a method for treating a cancer in a subject in need thereof,the method comprising: (a) administering a therapeutically effectiveamount of a first immunotherapy to the subject, wherein the firstimmunotherapy is a metabolic reprogramming agent; and (b) optionallyadministering a therapeutically effective amount of a secondimmunotherapy to the subject. In particular embodiments, the metabolicreprogramming agent is a glutamine antagonist. In particularembodiments, the metabolic reprogramming agent is a glutamine analogthat interferes with a glutamine metabolic pathway. In particularembodiments, the metabolic reprogramming agent is selected from thegroup consisting of acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), azaserine,and 6-diazo-5-oxo-norleucine (DON), and 5-diazo-4-oxo-L-norvaline(L-DONV). In particular embodiments, the metabolic reprogramming agentis a prodrug of a glutamine analog that interferes with a glutaminemetabolic pathway. In particular embodiments, at least one metabolicreprogramming agent is a prodrug of acivicin, azaserine, DON, andL-DONV.

In some aspects, a prodrug of a glutamine antagonist, or apharmaceutically acceptable salt or ester thereof has a structure offormula (I):

wherein: X is selected from the group consisting of a bond, —O—, and—(CH₂)_(n)—, wherein n is an integer selected from the group consistingof 1, 2, 3, 4, 5, 6, 7, and 8; R₁ is selected from the group consistingof H and a first prodrug-forming moiety capable of forming a salt or anester; and R₂ is H or a second prodrug-forming moiety capable of formingan amide linkage, a carbamate linkage, a phosphoramidate linkage or aphosphorodiamidate linkage with the nitrogen adjacent to R₂; R₂′ isselected from the group consisting of H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, or R₂ and R₂′ together form a ring structure comprising—C(═O)-G-C(═O)—, wherein G is selected from the group consisting ofC₁-C₈ alkylene, C heteroalkylene, C₅-C₈ cycloalkylene, C₆-C₁₂ arylene,C₅-C₁₄ heteroarylene, bivalent C₄-C₁₀ heterocycle, each of which can beoptionally substituted; or R₁ and R₂′ together form a 4- to 6-memberedheterocylic ring comprising the oxygen atom adjacent to R₁ and thenitrogen atom adjacent to R₂′; provided that the compound has at leastone prodrug-forming moiety selected from the group consisting of thefirst and the second prodrug-forming moieties.

As used herein, the term “amide linkage” comprises a structurerepresented by the formula:

wherein R_(v) is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substitutedaryl, aralkyl, substituted aralkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, alkylamine, substituted alkylamine, heteroaryl, andsubstituted heteroaryl.

As used herein, the term “carbamate linkage” comprises a structurerepresented by the formula:

wherein R_(w) is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substitutedaryl, aralkyl, substituted aralkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, alkylamine, substituted alkylamine, heteroaryl, andsubstituted heteroaryl.

As used herein, the term “phosphoramidate linkage” comprises a structurerepresented by the formula:

wherein R_(x) and R_(x)′ are each independently selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkylamine, substitutedalkylamine, heteroaryl, and substituted heteroaryl.

As used herein, the term “phosphorodiamidate linkage” comprises astructure represented by the formula:

wherein R_(y) and R_(z) are each independently selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl, alkenyl, substitutedalkenyl, cycloalkenyl, substituted cycloalkenyl, —(CR₃R₄)_(m)—Z,—(CR₃R₄)_(m)-Q-Z, aryl, substituted aryl, alkylamine, substitutedalkylamine, heteroaryl, substituted heteroaryl, and

In some embodiments, X is —CH₂—, and n is 1.

In other embodiments, X is —O—. In some embodiments, the prodrugcompound has both the first prodrug-forming moiety and the secondprodrug-forming moiety. In some embodiments, the glutamine analog is aglutamine antagonist, i.e., the prodrug is a prodrug of a glutamineanalog that antagonizes a glutamine pathway. Exemplary glutamineantagonists include, without limitation, 6-diazo-5-oxo-norleucine (DON),and aza-serine, and 5-diazo-4-oxo-L-norvaline (L-DONV).

In some embodiments, the presently disclosed subject matter provides aprodrug of DON. In some embodiments, the prodrug of DON has a structureof formula (I). In some embodiments, the presently disclosed subjectmatter provides a prodrug of L-DONV. In some embodiments, the prodrug ofL-DONV has a structure of formula (I). In some embodiments, thepresently disclosed subject matter provides a prodrug of azaserine. Insome embodiments, the prodrug of azaserine has a structure of formula(I).

In some embodiments, R₁ of formula (I) comprises a residue PRO₁ of theprodrug-forming moiety, which, together with a basic moiety and theterminal hydroxyl group forms a salt.

In some embodiments, R₁ of formula (I) comprises a residue PRO₁ of theprodrug-forming moiety, which, together with an alkyl group and theoxygen of an adjoining hydroxyl group forms an ester.

In some embodiments, R₁ of formula (I) comprises a residue PRO₁ of theprodrug-forming moiety, which, together with an alkyl group and thenitrogen adjoining the R₂′ group, forms an azlactone or an oxazolidone.

In some embodiments, R₁ of formula (I) is selected from the groupconsisting of H, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkenyl, substituted cycloalkenyl, tri(hydrocarbyl)ammonium, andtetra(hydrocarbyl)ammonium. Preferred alkyl group, cycloalkyl group,alkenyl group, alkynyl group, and cycloalkenyl group substituentsinclude alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.

In some embodiments, R₁ of formula (I) is not H. In some embodiments, R₁of formula (I) is not H when R₂ and R₂′ are H. In some embodiments, R₂and R₂′ of formula (I) are each H when and R₁ is not H.

In some embodiments, R₁ of formula (I) is selected from the groupconsisting of a C₁₋₆ straight-chain alkyl, a substituted C₁₋₆straight-chain alkyl, a C₁₋₆ branched alkyl, a substituted C₁₋₆ branchedalkyl, tri(C₁-C₈-alkyl)ammonium, tetra(C₁-C₈-alkyl)ammonium,triphenylammonium, tri(hydroxy-C₁-C₈-alkyl)ammonium, andtetra(hydroxy-C₁-C₈-alkyl)ammonium.

In some embodiments, R₁ of formula (I) is selected from the groupconsisting of methyl, ethyl, isopropyl, cyclopentyl, cyclohexyl, trimethyl ammonium, triethylammonium, tri(hydroxyethyl)ammonium,tripropylammonium, and tri(hydroxypropyl)ammonium. In some embodiments,R₁ of formula (I) is methyl. In some embodiments, R₁ of formula (I) isethyl. In some embodiments, R₁ of formula (I) is isopropyl.

In some embodiments, R₂ of formula (I) comprises a residue PRO₂ of thesecond prodrug-forming moiety, which, together with a carbonyl, oxycarbonyl, or phosphonyl group and the nitrogen of the adjoining NH,forms an amide, a carbamate, phosphoramidate, or phosphorodiamidatelinkage.

In some embodiments, R₂ of formula (I) comprises a moiety selected fromthe group consisting of an amino acid, an N-substituted amino acid, apeptide, a substituted peptide, a monocyclic ring, a substitutedmonocyclic ring, a bicyclic ring, a substituted bicyclic ring, a purinenucleoside, a substituted purine nucleoside, a pyrimidine nucleoside,and a substituted pyrimidine nucleoside.

In some embodiments, R₂ of formula (I) is selected from the groupconsisting of H, alkyl, —C(═O)—Ar, —C(═O)—Y—(CR₃R₄)_(m)—Ar,—C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆, —P(═O)(OR₇)_(n)(NHR₉)_(o),—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—R₈, —C(═O)—Y—(CR₃R₄)_(m)—Ar—O—R₈,—C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀, —C(═O)—O—R₉,—C(═O)—Y—(CR₃R₄)_(m)—Ar—O—C(═O)—Ar, and —C(═O)—Y—(CR₃R₄)_(m)—Ar—NR₅R₆;wherein: Y is —O— or a bond; m is an integer selected from the groupconsisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; each n and o is an integerfrom 0 to 2 provided that the sum of n and o is 2; R₃ and R₄ isindependently H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl, aryl orsubstituted aryl, —(CR₃R₄)_(m)—NR₅R₆, or

each R₅ and R₆ is independently H, alkyl, —C(═O)—(CR₃R₄)_(m),—C(═O)—(NR₅R₆), or —C(═O)—(CR₃R₄)_(m)—NR₅R₆; each R₇ is independentlyselected from the group consisting of H, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, —(CR₃R₄)_(m)—Z, —(CR₃R₄)_(m)-Q-Z, wherein Q is amonosaccharide, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, and wherein Z is

or wherein R7 together with the oxygen atom to which it is attachedforms a purine or pyrimidine nucleoside; each R₉ is independentlyselected from the group consisting of H, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substitutedcycloalkenyl, —(CR₃R₄)_(m)—Z, aryl, substituted aryl, heteroaryl,substituted heteroaryl, and

wherein R₁ and X are as defined above, provided that R₁ is not H; eachR₈ is independently alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, monosaccharide, acylated monosaccharide, aryl, substitutedaryl, heteroaryl, substituted heteroaryl; each R₁₀ is independentlyalkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,monosaccharide, acylated monosaccharide, aryl, substituted aryl,heteroaryl, substituted heteroaryl; and Ar is aryl, substituted aryl,heteroaryl, or substituted heteroaryl. It should be appreciated that inaddition to substitutions on the amino group of Z, one or moresubstitutions R₃, R₄, R₅, and/or R₆ can be made to the 5 or 6 memberedrings of Z.

The disclosure also provides the following particular embodimentsnumbered Embodiments I-LXI.

Embodiment I. A method of treating cancer in a subject, the methodcomprising administering to the subject in need thereof a compound, or apharmaceutically acceptable salt thereof, having formula (I):

wherein:

X is selected from the group consisting of a bond, —O—, and —(CH₂)_(n)—,wherein n is an integer selected from the group consisting of 1, 2, 3,4, 5, 6, 7, and 8;

R₁ is selected from the group consisting of C₁₋₆ alkyl and substitutedC₁₋₆ alkyl;

R₂ is an amino acid, an N-substituted amino acid, or—C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀;

R₂′ is selected from the group consisting of H, C₁-C₆ alkyl, andsubstituted C₁-C₆ alkyl;

each R₃ and R₄ are independently H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, aryl, substituted aryl, —(CR₃R₄)_(m)—NR₅R₆, or

m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, and 8;

R₅ and R₆ are independently H or alkyl; and

R₁₀ is selected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, monosaccharide, acylatedmonosaccharide, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl.

Embodiment II. The method of Embodiment I, wherein X is —CH₂—.

Embodiment III. The method of Embodiment I, wherein X is —O—.

Embodiment IV. The method of Embodiment I, wherein R₁ is selected fromthe group consisting of methyl, ethyl, isopropyl, cyclopentyl,cyclohexyl, trimethylammonium, triethylammonium,tri(hydroxyethyl)ammonium, tripropylammonium, andtri(hydroxypropyl)ammonium.

Embodiment V. The method of Embodiment I, wherein R₂ is selected fromthe group consisting of —C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆, and—C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀; wherein:

Y is —O— or a bond;

m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, and 8; and

each R₃ and R₄ is independently H, C₁-C₆ alkyl or substituted C₁-C₆alkyl, aryl or substituted aryl;

R₁₀ is selected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,and substituted heteroaryl.

Embodiment VI. The method of Embodiment V, wherein:

Y is a bond;

m is 1;

R₅ and R₆ are each H.

Embodiment VII. The method of Embodiment I, wherein R₂ is an amino acid.

Embodiment VIII. The method of Embodiment VII, wherein the amino acid istryptophan.

Embodiment IX. The method of Embodiment I, wherein R₂ is a N-acyl aminoacid.

Embodiment X. The method of Embodiment IX, wherein the amino acid istryptophan.

Embodiment XI. The method of Embodiment I, wherein the compound havingformula (I) is a compound having formula (IIA):

wherein:

R₁ is selected from the group consisting of H and C₁₋₆ alkyl;

R₁₁ is selected from the group consisting of H, methyl, isopropyl,sec-butyl, CH₂CH(CH₃)₂, benzyl, p-hydroxybenzyl CH₂OH, CH(OH)CH₃,CH₂-3-indoyl, CH₂COOH, CH₂CH₂COOH, —CH₂C(O)NH₂, CH₂CH₂C(O)NH₂, CH₂SH,CH₂CH₂SCH₃, (CH₂)₄NH₂, (CH₂)₃NHC(═NH)NH₂, and CH₂-3-imidazoyl;

R₁₂ is selected from the group consisting of H, C₁₋₄ alkyl, and—C(═O)R₁₃; and

R₁₃ is C₁₋₄ alkyl.

Embodiment XII. The method of Embodiment I, wherein the compound havingformula (I) is a compound having formula (IIB):

wherein:

R₁ is selected from the group consisting of H and C₁₋₆ alkyl;

R₁₁ is selected from the group consisting of H, methyl, isopropyl,sec-butyl, CH₂CH(CH₃)₂, benzyl, p-hydroxybenzyl CH₂OH, CH(OH)CH₃,CH₂-3-indoyl, CH₂COOH, CH₂CH₂COOH, —CH₂C(O)NH₂, CH₂CH₂C(O)NH₂, CH₂SH,CH₂CH₂SCH₃, (CH₂)₄NH₂, (CH₂)₃NHC(═NH)NH₂, and CH₂-3-imidazoyl;

R₁₂ is selected from the group consisting of H, C₁₋₄ alkyl, and—C(═O)R₁₃; and

R₁₃ is C₁₋₄ alkyl.

Embodiment XIII. The method of Embodiments XI or XII, wherein:

R₁ is C₁₋₄ alkyl;

R₁₁ is selected from the group consisting of isopropyl, sec-butyl,CH₂CH(CH₃)₂, and CH₂-3-indoyl;

R₁₂ is selected from the group consisting of H and —C(═O)R₁₃; and

R₁₃ is C₁₋₄ alkyl.

Embodiment XIV. The method of Embodiment I, wherein the compound offormula (I) is selected from the group consisting of:

Embodiment XV. The method of Embodiment XIV, wherein the compound is:

Embodiment XVI. The method of Embodiment I, wherein the compound havingformula (I) is a compound having formula (III):

wherein:

R₁ is selected from the group consisting of H and C₁₋₆ alkyl;

R₃ and R₄ are independently selected from the group consisting of H,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, aryl, and substituted aryl; and

R₁₀ is C₁₋₆ alkyl.

Embodiment XVII. The method of Embodiment XVI, wherein:

R₃ is selected from the group consisting of C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, aryl, and substituted aryl; and

R₄ is H.

Embodiment XVIII. The method of Embodiment XVII, wherein:

R₃ is H; and

R₄ is selected from the group consisting of C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, aryl, and substituted aryl.

Embodiment XIX. The method of Embodiment I, wherein the compound offormula (I) is selected from the group consisting of:

Embodiment XX. The method of Embodiment XIX, wherein the compoundselected from the group consisting of:

Embodiment XXI. A method of treating cancer in a subject, the methodcomprising administering to the subject in need thereof a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of:

Embodiment XXII. The method of any one of Embodiments I-XXI furthercomprising simultaneously or sequentially administering atherapeutically effective amount of an immunotherapeutic agent to thesubject.

Embodiment XXIII. The method of Embodiment XXII, wherein theimmunotherapeutic agent is an immune checkpoint blockade therapy.

Embodiment XXIV. The method of Embodiment XXIII, wherein the immunecheckpoint blockade therapy is selected from the group consisting ofPD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, LAG3antagonists, and B7-H₃ antagonists, and combinations thereof.

Embodiment XXV. The method of Embodiment XXII, wherein theimmunotherapeutic agent is an adoptive cellular therapy.

Embodiment XXVI. The method of Embodiment XXII, wherein theimmunotherapeutic agent is marrow-infiltrating lymphocytes (MILs).

Embodiment XXVII. The method of Embodiment XXII, wherein theimmunotherapeutic agent is an adenosine A2aR inhibitor.

Embodiment XXVIII. The method of Embodiment XXII, wherein theimmunotherapeutic agent is a tumor vaccine.

Embodiment XXIX. The method of Embodiment XXII, wherein theimmunotherapeutic agent is a passive immunotherapy antibody.

Embodiment XXX. The method of Embodiment XXIX, wherein the passiveimmunotherapy antibody is selected from the group consisting ofbevacizumab, cetuximab, rituximab, trastuzumab, alemtuzumab, ibritumomabtiuxetan, and panitumumab, and combinations thereof.

Embodiment XXXI. The method of any one of Embodiments I-XXX wherein thecancer is:

(i) a cancer of the central nervous system;

(ii) a cancer that is associated with transplant and/orimmunosuppression;

(iii) a cancer that is refractory to chemotherapy;

(iv) a cancer that is refractory to photodynamic therapy;

(v) a cancer that is refractory to proton therapy;

(vi) a cancer that is refractory to radiotherapy; and

(vii) a cancer that is refractory to surgery.

Embodiment XXXII. The method of any one of Embodiments I-XXX, whereinthe cancer is a newly diagnosed, recurrent, and/or refractory cancerselected from the group consisting of celnasopharyngeal cancer, synovialcancer, hepatocellular cancer, renal cancer, cancer of connectivetissues, melanoma, lung cancer, bowel cancer, colon cancer, rectalcancer, colorectal cancer, brain cancer, throat cancer, oral cancer,liver cancer, bone cancer, pancreatic cancer, choriocarcinoma,gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma,neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenalcancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer,brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cordtumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma,cancer of unknown primary site, carcinoid, carcinoid of gastrointestinaltract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer,colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer,head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, livercancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicularcancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skincancer, mesothelioma, multiple myeloma, ovarian cancer, endocrinepancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer,penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma,small intestine cancer, stomach cancer, thymus cancer, thyroid cancer,trophoblastic cancer, hydatidiform mole, uterine cancer, endometrialcancer, vagina cancer, vulva cancer, acoustic neuroma, mycosisfungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer,heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer,palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer,pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

Embodiment XXXIII A method of preventing a relapse or reducing theincidence of relapse of a cancer subject in remission, the methodcomprising administering to the subject in need thereof a compound, or apharmaceutically acceptable salt thereof, having formula (I):

wherein:

X is selected from the group consisting of a bond, —O—, and —(CH₂)_(n)—,wherein n is an integer selected from the group consisting of 1, 2, 3,4, 5, 6, 7, and 8;

R₁ is selected from the group consisting of C₁₋₆ alkyl and substitutedC₁₋₆ alkyl;

R₂ is an amino acid, an N-substituted amino acid, or—C(═O)—O—(CR₃R₄)_(m)—O—C(═O)—R₁₀;

R₂′ is selected from the group consisting of H, C₁-C₆ alkyl, andsubstituted C₁-C₆ alkyl;

each R₃ and R₄ are independently H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, aryl, substituted aryl, —(CR₃R₄)_(m)—NR₅R₆, or

m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, and 8;

R₅ and R₆ are independently H or alkyl; and

R₁₀ is selected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, monosaccharide, acylatedmonosaccharide, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl.

Embodiment XXXIV. The method of Embodiment XXXIII, wherein X is —CH₂—.

Embodiment XXXV. The method of Embodiment XXXIII, wherein X is —O—.

Embodiment XXXVI. The method of any one of Embodiments XXXIII-XXXV,wherein R₁ is selected from the group consisting of methyl, ethyl,isopropyl, cyclopentyl, cyclohexyl, trimethylammonium, triethylammonium,tri(hydroxyethyl)ammonium, tripropylammonium, andtri(hydroxypropyl)ammonium.

Embodiment XXXVII. The method of any one of Embodiments XXXIII-XXVI,wherein R₂ is selected from the group consisting of—C(═O)—Y—(CR₃R₄)_(m)—NR₅R₆, and —C(═O)—O—(CR₃R₄)_(m)-O—C(═O)—R₁₀;

wherein:

Y is —O— or a bond;

m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, and 8; and

each R₃ and R₄ is independently H, C₁-C₆ alkyl or substituted C₁-C₆alkyl, aryl or substituted aryl;

R₁₀ is selected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,and substituted heteroaryl.

Embodiment XXXVIII. The method of Embodiment XXXVII, wherein:

Y is a bond;

m is 1;

R₅ and R₆ are each H.

Embodiment XXXIX. The method of any one of Embodiments XXXIII-XXVI,wherein R₂ is an amino acid.

Embodiment XL. The method of Embodiment XXXIX, wherein the amino acid istryptophan.

Embodiment XLI. The method of any one of Embodiment XXXIII-XXVI, whereinR₂ is a N-acyl amino acid.

Embodiment XLII. The method of Embodiment XLI, wherein the amino acid istryptophan.

Embodiment XLIII. The method of Embodiment XXXIII, or a pharmaceuticallyacceptable salt thereof, having formula (IIA):

wherein:

R₁ is selected from the group consisting of H and C₁₋₆ alkyl;

R₁₁ is selected from the group consisting of H, methyl, isopropyl,sec-butyl, CH₂CH(CH₃)₂, benzyl, p-hydroxybenzyl CH₂OH, CH(OH)CH₃,CH₂-3-indoyl, CH₂COOH, CH₂CH₂COOH, —CH₂C(O)NH₂, CH₂CH₂C(O)NH₂, CH₂SH,CH₂CH₂SCH₃, (CH₂)₄NH₂, (CH₂)₃NHC(═NH)NH₂, and CH₂-3-imidazoyl;

R₁₂ is selected from the group consisting of H, C₁₋₄ alkyl, and—C(═O)R₁₃; and

R₁₃ is C₁₋₄ alkyl.

Embodiment XLIV. The method of Embodiment XXXIII, wherein the compoundhaving formula (I) is a compound having formula (IIB):

wherein:

R₁ is selected from the group consisting of H and C₁₋₆ alkyl;

R₁₁ is selected from the group consisting of H, methyl, isopropyl,sec-butyl, CH₂CH(CH₃)₂, benzyl, p-hydroxybenzyl CH₂OH, CH(OH)CH₃,CH₂-3-indoyl, CH₂COOH, CH₂CH₂COOH, —CH₂C(O)NH₂, CH₂CH₂C(O)NH₂, CH₂SH,CH₂CH₂SCH₃, (CH₂)₄NH₂, (CH₂)₃NHC(·NH)NH₂, and CH₂-3-imidazoyl;

R₁₂ is selected from the group consisting of H, C₁₋₄ alkyl, and—C(═O)R₁₃; and

R₁₃ is C₁₋₄ alkyl.

Embodiment XLV. The method of Embodiments XLIII or XLIV, wherein:

R₁ is C₁₋₄ alkyl;

R₁₁ is selected from the group consisting of isopropyl, sec-butyl,CH₂CH(CH₃)₂, and CH₂-3-indoyl;

R₁₂ is selected from the group consisting of H and —C(═O)R₁₃; and

R₁₃ is C₁₋₄ alkyl.

Embodiment XLVI. The method of Embodiment XXXIII, wherein the compoundof formula (I) is selected from the group consisting of:

Embodiment XLVII. The method of Embodiment XLVI wherein the compound is:

Embodiment XLVIII. The method of Embodiment XXXIII, wherein the compoundhaving formula (I) is a compound having formula (III):

wherein:

R₁ is selected from the group consisting of H and C₁₋₆ alkyl;

R₃ and R₄ are independently selected from the group consisting of H,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, aryl, and substituted aryl; and

R₁₀ is C₁₋₆ alkyl.

Embodiment XLIX. The method of Embodiment XXXIII, wherein the compoundof formula (I) is selected from the group consisting of:

Embodiment L. The method of Embodiment XLIX selected from the groupconsisting of:

Embodiment LI. A method of preventing a relapse or reducing theincidence of relapse of a cancer subject in remission, the methodcomprising administering to the subject in need thereof a compound, or apharmaceutically acceptable salt thereof, selected from the groupconsisting of:

Embodiment LII. The method any one of Embodiments XXXIII-LI, wherein thecompound is:

(i) administered to the subject post transplant;

(ii) administered to the subject post chemotherapy;

(iii) administered to the subject post immunotherapy;

(iv) administered to the subject post photodynamic therapy;

(v) administered to the subject post proton therapy;

(vi) administered to the subject post radiotherapy;

(vii) administered to the subject post surgery; or

(viii) after relapse of a cancer of a subject, and combinations thereof.

Embodiment LIII. The method of any one of Embodiments XXXIII-LII furthercomprising simultaneously or sequentially administering atherapeutically effective amount of an immunotherapeutic agent to thesubject.

Embodiment LIV. The method of Embodiment LIII, wherein theimmunotherapeutic agent is an immune checkpoint blockade therapy.

Embodiment LV. The method of Embodiment LIV, wherein the immunecheckpoint blockade therapy is selected from the group consisting ofPD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, LAG3antagonists, and B7-H3 antagonists, and combinations thereof.

Embodiment LVI. The method of Embodiment LIII, wherein theimmunotherapeutic agent is an adoptive cellular therapy.

Embodiment LVII. The method of Embodiment LIII, wherein theimmunotherapeutic agent is marrow-infiltrating lymphocytes (MILs).

Embodiment LVIII. The method of Embodiment LIII, wherein theimmunotherapeutic agent is an adenosine A2aR inhibitor.

Embodiment LIX. The method of Embodiment LIII, wherein theimmunotherapeutic agent is a tumor vaccine.

Embodiment LX. The method of Embodiment LIII, wherein theimmunotherapeutic agent is a passive immunotherapy antibody.

Embodiment LXI. The method of Embodiment LX, wherein the passiveimmunotherapy antibody is selected from the group consisting ofbevacizumab, cetuximab, rituximab, trastuzumab, alemtuzumab, ibritumomabtiuxetan, and panitumumab, and combinations thereof.

The disclosure also provides the following particular embodimentsnumbered Embodiments 1-20.

Embodiment 1. A method for treating a cancer in a subject in needthereof, the method comprising:

(a) administering a therapeutically effective amount of a firstimmunotherapy to the subject, wherein the first immunotherapy is ametabolic reprogramming agent that decreases glutamine metabolicactivity; and

(b) optionally administering a therapeutically effective amount of asecond immunotherapy to the subject.

Embodiment 2. The method of Embodiment 1, wherein the metabolicreprogramming agent is a glutamine antagonist.

Embodiment 3. The method of Embodiment 1, wherein the metabolicreprogramming agent is a glutamine analog that interferes with aglutamine metabolic pathway.

Embodiment 4. The method of Embodiment 1, wherein the metabolicreprogramming agent is selected from the group consisting of acivicin(L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleaceticacid), azaserine, and 6-diazo-5-oxo-norleucine (DON), and5-diazo-4-oxo-L-norvaline (L-DONV).

Embodiment 5. The method of Embodiment 1, wherein the metabolicreprogramming agent is a prodrug of a glutamine analog that interfereswith a glutamine metabolic pathway.

Embodiment 6. The method of Embodiment 1, wherein the at least onemetabolic reprogramming agent is a prodrug of acivicin, azaserine, DON,and L-DONV.

Embodiment 7. The method of Embodiment 1, further comprisingsimultaneously or sequentially administering a therapeutically effectiveamount of the second immunotherapy to the subject, wherein the secondimmunotherapy is an immune checkpoint blockade therapy.

Embodiment 8. The method of Embodiment 7, wherein the immune checkpointblockade therapy is selected from the group consisting of PD-1antagonists, PD-L1 antagonists, CTLA-4 antagonists, LAG3 antagonists,B7-H₃ antagonists, and combinations thereof.

Embodiment 9. The method of Embodiment 1, further comprisingsimultaneously or sequentially administering a therapeutically effectiveamount of the second immunotherapy to the subject, wherein the secondimmunotherapy is an adoptive cellular therapy.

Embodiment 10. The method of Embodiment 1, further comprisingsimultaneously or sequentially administering a therapeutically effectiveamount of the second immunotherapy to the subject, wherein the secondimmunotherapy is marrow-infiltrating lymphocytes (MILs).

Embodiment 11. The method of Embodiment 1, further comprisingsimultaneously or sequentially administering a therapeutically effectiveamount of the second immunotherapy to the subject, wherein the secondimmunotherapy is an adenosine A2aR blockade.

Embodiment 12. The method of Embodiment 1, further comprisingsimultaneously or sequentially administering a therapeutically effectiveamount of the second immunotherapy to the subject, wherein the secondimmunotherapy is a tumor vaccine.

Embodiment 13. The method of Embodiment 1, further comprisingsimultaneously or sequentially administering a therapeutically effectiveamount of the second immunotherapy to the subject, wherein the secondimmunotherapy is a passive immunotherapy antibody.

Embodiment 14. The method of Embodiment 14, wherein the passiveimmunotherapy antibody is selected from the group consisting ofbevacizumab, cetuximab, rituximab, trastuzumab, alemtuzumab, ibritumomabtiuxetan, panitumumab, and combinations thereof.

Embodiment 15. The method of Embodiment 1, further comprisingsimultaneously or sequentially administering to the subject atherapeutically effective amount of a cancer therapy selected from thegroup consisting of: (i) chemotherapy; (ii) photodynamic therapy; (iii)proton therapy; (iv) radiotherapy; (v) surgery; and combinationsthereof.

Embodiment 16. The method of Embodiment 1, wherein the firstimmunotherapy, and the second immunotherapy if administered, is/areadministered to the subject in the absence of a cancer therapy selectedfrom the group consisting of: (i) chemotherapy; (ii) photodynamictherapy; (iii) proton therapy; (iv) radiotherapy; (v) surgery; andcombinations thereof.

Embodiment 17. The method of Embodiment 1, wherein the cancer is:

(i) a cancer of the central nervous system;

(ii) a cancer that is associated with transplant and/orimmunosuppression;

(iii) a cancer that is refractory to chemotherapy;

(iv) a cancer that is refractory to photodynamic therapy;

(v) a cancer that is refractory to proton therapy;

(vi) a cancer that is refractory to radiotherapy;

(vii) a cancer that is refractory to surgery, or

(vii) a cancer that has relapsed.

Embodiment 18. The method of Embodiment 1, wherein the cancer is a newlydiagnosed, recurrent, and/or refractory cancer selected from the groupconsisting of celnasopharyngeal cancer, synovial cancer, hepatocellularcancer, renal cancer, cancer of connective tissues, melanoma, lungcancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer,brain cancer, throat cancer, oral cancer, liver cancer, bone cancer,pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma,prolactinoma, T-cell leukemia/lymphoma, neuroma, von Hippel-Lindaudisease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bileduct cancer, bladder cancer, ureter cancer, oligodendroglioma,neuroblastoma, meningioma, spinal cord tumor, bone cancer,osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknownprimary site, carcinoid, carcinoid of gastrointestinal tract,fibrosarcoma, breast cancer, Paget's disease, cervical cancer,colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer,head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, livercancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicularcancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skincancer, mesothelioma, multiple myeloma, ovarian cancer, endocrinepancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer,penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma,small intestine cancer, stomach cancer, thymus cancer, thyroid cancer,trophoblastic cancer, hydatidiform mole, uterine cancer, endometrialcancer, vagina cancer, vulva cancer, acoustic neuroma, mycosisfungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer,heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer,palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer,pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

Embodiment 19. A method of preventing a relapse or reducing theincidence of relapse of a cancer subject in remission, the methodcomprising administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent, wherein the metabolicreprogramming agent is selected from the group consisting of acivicin(L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleaceticacid), azaserine, and 6-diazo-5-oxo-norleucine (DON), and5-diazo-4-oxo-L-norvaline (L-DONV), and prodrugs thereof.

Embodiment 20. The method of Embodiment 20, wherein the metabolicreprogramming agent is:

(i) administered to the subject post transplant;

(ii) administered to the subject post chemotherapy;

(iii) administered to the subject post immunotherapy;

(iv) administered to the subject post photodynamic therapy;

(v) administered to the subject post proton therapy;

(vi) administered to the subject post radiotherapy;

(vii) administered to the subject post surgery; or

(vii) administered to a subject after relapse of a cancer, andcombinations thereof.

Structures of representative DON prodrugs are provided in Table 1.

TABLE 1 Structures of Representative DON Prodrugs IOCB No./ Compound No.Structure MW Compound 1 (DON)

171.15 Compound 3

213.24 Compound 4

445.41 Compound 6

391.38 Compound 7

564.53 Compound 9

326.39 Compound 11

439.55 Compound 13

369.18 Compound 14a ^(#)

385.41 Compound 14b ^(#) (or 5c)

Compound 15

371.39 Compound 17

375.33 Compound 20

199.21 Compound 22

270.28 Compound 23

343.42 Compound 25

312.36 Compound 26

385.50 Compound 28

425.52 Compound 29

329.31 Compound 30

343.33 Compound 31

357.37 Compound 32

371.39 Compound 34

385.42 Compound 35

327.25 Compound 36

355.30 Compound 38

399.45 Compound 38a

Compound 40

413.47 Compound 42

371.39 Compound 44

2.44 Compound 47

447.49 Compound 49

357.36 Compound 51

618.69 Compound 52

660.73 Compound 56

469.54 Compound 57

511.58 Compound 59

511.48 Compound 60

464.19 Compound 60a

A

618.54 B

602.54 C

530.47 D

334.38 E

484.51 F

525.51 G

509.51 LTP 073

255.23 JAM0351

693.66 JAM0359

679.63 ^(#) A diastereomeric mixture of isopropyl(2S)-6-diazo-5-oxo-2-(((1-(pivaloyloxy)ethoxy)carbonyl)amino)hexanoatewas prepared and separated by column chromatography to give isopropyl(S)-6-diazo-5-oxo-2-((((S)-1-(pivaloyloxy)ethoxy)carbonyl)amino)hexanoateand isopropyl(S)-6-diazo-5-oxo-2-((((R)-1-(pivaloyloxy)ethoxy)carbonyl)amino)hexanoate.The S,S-isomer was arbitrarily designated compound 14a, and theS,R-isomer was arbitrarily designated compound 14b. The actualstereochemistry of the acetal methyl group was not determined. Thediastereoisomer that was arbitrarily designated compound 14b was used inthe biological studies described herein. See PCT/US2016/044767 (WO2017/023774 A1), which is fully incorporated by reference herein.

1. Checkpoint Blockade

Aspects of the presently disclosed subject matter involve the use ofmetabolic reprogramming agents (e.g., DON, DON prodrugs, etc.) in acombination immunotherapy together with checkpoint blockade modulators,for example, to enhance checkpoint blockade therapies for the treatmentof cancer. In some aspects, the presently disclosed subject matterinvolves the use of metabolic reprogramming agents (e.g., DON, DONprodrugs, etc.) in combination immunotherapy together with A2aRblockade, and optionally in combination with a third immunotherapy, afourth immunotherapy, and/or a fifth immunotherapy, such as tumorvaccines, A2aR blockade, and/or adoptive cell therapy.

Accordingly, in some embodiments, the method of treating cancer furtherincludes simultaneously or sequentially administering a therapeuticallyeffective amount of the second immunotherapy to the subject, wherein thesecond immunotherapy is an immune checkpoint blockade modulator. As usedherein, the term “immune checkpoint modulator” refers to an agent thattotally or partially reduces, inhibits, interferes with, activates, ormodulates one or more checkpoint proteins (i.e., an immune checkpointreceptor or a ligand for the immune checkpoint receptor).

Examples of immune checkpoint modulators of use herein include, but arenot limited to, small organic molecules (e.g., haptens) or smallinorganic molecules; saccharides; oligosaccharides; polysaccharides; abiological macromolecule selected from the group consisting of peptides(e.g., aptides), proteins, peptide analogs and derivatives;peptidomimetics; nucleic acids selected from the group consisting ofmiRNAs, siRNAs, shRNAs, antisense nucleic acids, such as antisense RNAs,ribozymes, and aptamers; an extract made from biological materialsselected from the group consisting of bacteria, plants, fungi, animalcells, and animal tissues; naturally occurring or syntheticcompositions; and any combination thereof. Other examples of immunecheckpoint modulators include orthosteric inhibitors, allostericregulators, interfacial binders, and molecular analogues of substratesthat act as competitive inhibitors.

Specific examples of immune checkpoint modulators include, withoutlimitation, PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists,Lag-3 antagonists, CD137 antagonists, KIR antagonists, Tim3 antagonists,Ox40 agonists, B7-H₃ antagonists, and combinations thereof.

Exemplary CTLA-4 antagonists include, without limitation, ipilimumab,tremelimumab and combinations thereof. Anti-CTLA-4 antibodies arecurrently undergoing clinical trials for the treatment of melanoma.

Examplary Lag-3 antagonists include, without limitation, BMS-986016 andIMP321.

Exemplary CD137 antagonists include, without limitation, CD137-specificantibody, peptide, organic small molecule, antisense oligonuclotide,siRNA, antisense expression vector or recombinant virus. In someembodiments, the CD137-specific antibody is clone BBK-2 or clone 4B4-1,as described in WIPO International Application Publication No.WO200405513A2, which is incorporated herein by reference in itsentirety.

T-cell immunoglobulin and mucin domain 3 (TIM3) antagonists (e.g.,anti-TIM3 antibodies) have been described for use as immunotherapy (see,e.g., Ngiow et al. 2011). Exemplary Tim3 antagonists include, withoutlimitation, anti-TIM3 monoclonal antibodies, for example, as describedin the poster presentation by Jun et al. “Generation of antagonisticanti-TIM-3 and anti-LAG-3 monoclonal antibodies for potential novelimmunotherapy combinations”, available on the world wide web athttp://www.tesarobio.com/documents/2014AACRposterLB266.pdf, which isincorporated herein by reference.

Ox40 agonists are described by Linch et al., “OX40 Agonists andCombination Immunotherapy: Putting the Pedal to the Metal” Front Oncol5:34; 2015, which is incorporated herein by reference in its entirety.Exemplary Ox40 agonists include, without limitation, anti-Ox40 agonistsantibodies. Other exemplary Ox40 agonists include, without limitation,OX86 and Fc-OX40L.

Exemplary B7-H₃ antagonists include, without limitation, MGA271.

Exemplary PD-L1 antagonists include, without limitation,BMS-936559/MDX-1105, MEDI4736, MPDL3280A, MPDL3280A, MSB0010718C, andcombinations thereof. PD-L1 antagonists are currently undergoingclinical trials, for example, for the treatment of melanoma, non-smallcell lung cancer, renal cell carcinoma, and ovarian cancer.

PD-1 antagonists have been reviewed (see, e.g., Dolan and Gupta 2014).

Exemplary PD-1 antagonists of use herein include, without limitation,AMP-224, AMP-554, nivolumab, pembrolizumab, pidilizumab, andcombinations thereof.

In some embodiments, the PD-1 antagonists comprise anti-PD-1 antibodies.Exemplary anti-PD-1 antibodies include, without limitation,atezolizumab, nivolumab, pembrolizumab, pidilizumab, and combinationsthereof. PD-1 antagonists are currently undergoing clinical trials, forexample, for the treatment of colorectal cancer, gastric cancer,melanoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer,and renal cell carcinoma.

In particular embodiments, the presently disclosed subject matterprovides a method of treating an advanced solid tumor, the methodcomprising: (a) administering to the subject a therapeutically effectiveamount of BMS-936559; and (b) sequentially or simultaneouslyadministering to the subject a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating an advanced solid tumor, the methodcomprising: (a) administering to the subject a therapeutically effectiveamount of MEDI4736; and (b) sequentially or simultaneously administeringto the subject a therapeutically effective amount of a metabolicreprogramming agent that decreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating melanoma, the method comprising: (a)administering to the subject a therapeutically effective amount ofMPDL3280A in combination with vemurafenib; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating melanoma, the method comprising: (a)administering to the subject a therapeutically effective amount ofMEDI4736 in combination with dabrafenib and trametinib; and (b)sequentially or simultaneously administering to the subject atherapeutically effective amount of a metabolic reprogramming agent thatdecreases glutamine metabolism. In particular embodiments, the presentlydisclosed subject matter provides a method of treating melanoma, themethod comprising: (a) administering to the subject a therapeuticallyeffective amount of MEDI4736 in combination with trametinib; and (b)sequentially or simultaneously administering to the subject atherapeutically effective amount of a metabolic reprogramming agent thatdecreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating non-small cell lung cancer, the methodcomprising: (a) administering to the subject a therapeutically effectiveamount of MPDL3280A; and (b) sequentially or simultaneouslyadministering to the subject a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism. Insome embodiments, MPDL3280A is administered in combination witherlotinib. In particular embodiments, the presently disclosed subjectmatter provides a method of treating non-small cell lung cancer, themethod comprising: (a) administering to the subject a therapeuticallyeffective amount of MEDI4736; and (b) sequentially or simultaneouslyadministering to the subject a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism. Insome embodiments, MEDI4736 is administered in combination withtremelimumab.

In particular embodiments, the presently disclosed subject matterprovides a method of treating renal cell carcinoma, the methodcomprising: (a) administering to the subject a therapeutically effectiveamount of MPDL3280A; and (b) sequentially or simultaneouslyadministering to the subject a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism. Insome embodiments, MPDL3280A is administered in combination withbevacizumab.

In particular embodiments, the presently disclosed subject matterprovides a method of treating a solid or hematological malignancy, themethod comprising: (a) administering to the subject a therapeuticallyeffective amount of MPDL3280A; and (b) sequentially or simultaneouslyadministering to the subject a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating a solid tumor, the method comprising: (a)administering to the subject a therapeutically effective amount ofMPDL3280A; and (b) sequentially or simultaneously administering to thesubject a therapeutically effective amount of a metabolic reprogrammingagent that decreases glutamine metabolism. In some embodiments,MPDL3280A is administered in combination with bevacizumab and/orchemotherapy. In some embodiments, MPDL3280A is administered incombination with cobimetinib.

In particular embodiments, the presently disclosed subject matterprovides a method of treating a solid tumor, the method comprising: (a)administering to the subject a therapeutically effective amount ofMEDI4736; and (b) sequentially or simultaneously administering to thesubject a therapeutically effective amount of a metabolic reprogrammingagent that decreases glutamine metabolism. In some embodiments, MEDI4736is administered in combination with tremelimumab.

In particular embodiments, the presently disclosed subject matterprovides a method of treating a solid tumor, the method comprising: (a)administering to the subject a therapeutically effective amount ofMSB0010718C; and (b) sequentially or simultaneously administering to thesubject a therapeutically effective amount of a metabolic reprogrammingagent that decreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating advanced cancer, the method comprising:(a) administering to the subject a therapeutically effective amount ofAMP-224; and (b) sequentially or simultaneously administering to thesubject a therapeutically effective amount of a metabolic reprogrammingagent that decreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating an advanced solid tumor, the methodcomprising: (a) administering to the subject a therapeutically effectiveamount nivolumab in combination with iliolumbar (anti-KIR); and (b)sequentially or simultaneously administering to the subject atherapeutically effective amount of a metabolic reprogramming agent thatdecreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating a castration-resistant prostate cancer,hepatocellular carcinoma, melanoma, non-small cell lung cancer, or renalcell carcinoma, the method comprising: (a) administering to the subjecta therapeutically effective amount of nivolumab; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating colon cancer, gastric cancer, head andneck cancer, Hodgkin lymphoma, melanoma, myeloma, myelodysplasticsyndrome, non-Hodkin lymphoma, non-small cell lung cancer, solid tumors,or triple-negative breast cancer, the method comprising: (a)administering to the subject a therapeutically effective amount ofpembrolizumab; and (b) sequentially or simultaneously administering tothe subject a therapeutically effective amount of a metabolicreprogramming agent that decreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating gastric cancer, pancreatic cancer,small-cell lung cancer, glioblastoma, or triple-negative breast cancer,the method comprising: (a) administering to the subject atherapeutically effective amount of nivolumab in combination withipilimumab; and (b) sequentially or simultaneously administering to thesubject a therapeutically effective amount of a metabolic reprogrammingagent that decreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating a malignant glioma, the method comprising:(a) administering to the subject a therapeutically effective amount ofpidilizumab; and (b) sequentially or simultaneously administering to thesubject a therapeutically effective amount of a metabolic reprogrammingagent that decreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating pancreatic cancer, the method comprising:(a) administering to the subject a therapeutically effective amount ofpidilizumab in combination with gemcitabine; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating renal cell carcinoma, the methodcomprising: (a) administering to the subject a therapeutically effectiveamount of pidilizumab in combination with dendritic cell/RCC fusion cellvaccine; and (b) sequentially or simultaneously administering to thesubject a therapeutically effective amount of a metabolic reprogrammingagent that decreases glutamine metabolism.

2. Adoptive Cellular Therapy

Adoptive cell therapies (ACT) are a useful approach for treating cancer.Adoptive cell transfer refers to the passive transfer of ex vivo growncells, often immune-derived cells, into a host with the aim oftransferring the immunologic functionality and characteristics of thetransplant. Adoptive cell transfer can be autologous, as is common inadoptive T-cell therapies, or allogeneic. The adoptive transfer ofautologous tumor infiltrating lymphocytes (TILs) or geneticallyre-directed peripheral blood mononuclear cells has been used tosuccessfully treat patients with advanced solid tumors such as melanomaas well as patients with CD19-expressing hematologic malignancies.Exemplary cell types for use in ACT include, without limitation, T-cells(e.g., CD8+ cells, CD4+ cells, etc.), NK-cells, delta-gamma T-cells,regulatory T-cells and peripheral blood mononuclear cells. Such cellscan be unmodified such as in TIL therapy or genetically modified. Oneway to achieve genetic targeting of T-cells to tumor specific targets isthe transfer of a T-cell receptor with known specificity (TCR therapy)and with matched human leukocyte antigen (HLA, known as majorhistocompatibility complex in rodents) type. Another way is themodification of cells with artificial molecules such as chimeric antigenreceptors (CAR), commonly known as CAR-T cell therapy. For example,single chain antibodies can be used and CARs can also incorporateco-stimulatory domains.

Aspects of the presently disclosed subject matter involve the use ofmetabolic reprogramming agents (e.g., DON, DON prodrugs, etc.) incombination immunotherapy together with adoptive cellular therapy, forexample, to enhance adoptive cellular therapy for the treatment ofcancer. In some aspects, the presently disclosed subject matter involvesthe use of metabolic reprogramming agents (e.g., DON, DON prodrugs,etc.) in combination immunotherapy together with adoptive cellulartherapy, and optionally in combination with a third immunotherapy, afourth immunotherapy, and/or a fifth immunotherapy, such as tumorvaccines, A2aR blockade, and/or checkpoint blockade.

Accordingly, in some embodiments, the method of treating cancer furtherincludes simultaneously or sequentially administering a therapeuticallyeffective amount of the second immunotherapy to the subject, wherein thesecond immunotherapy is an adoptive cellular therapy. In someembodiments, the adoptive cellular therapy is selected from the groupconsisting of T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gammaT-cells, marrow infiltrating lymphocytes (MILs), regulatory T-cells, andperipheral blood mononuclear cells. In some embodiments, the adoptivecellular therapy comprises a tumor infiltrating lymphocyte (TIL). Insome embodiments, the adoptive cellular therapy comprises a T-cellreceptor modified lymphocyte. In some embodiments, the adoptive cellulartherapy comprises a chimeric antigen receptor modified lymphocyte. Insome embodiments, the adoptive cellular therapy comprises a chimericantigen receptor T (CAR-T) cell. In some embodiments, the adoptivecellular therapy comprises marrow infiltrating lymphocytes (MILs). Insome embodiments, the method of treating cancer further includessimultaneously or sequentially administering a therapeutically effectiveamount of the second immunotherapy to the subject, wherein the secondimmunotherapy is marrow-infiltrating lymphocytes (MILs).

3. Adenosine A2Ar Blockade

Adenosine A2a receptor (A2aR) blockade has been reviewed and is reportedto enhance tumor vaccines, checkpoint blockade and adoptive T celltherapy (see, e.g., Powell et al. 2015). Accordingly, aspects of thepresently disclosed subject matter involves the use of metabolicreprogramming agents (e.g., DON, DON prodrugs, etc.) in combinationimmunotherapy together with adenosine A2a receptor (A2aR) blockade, forexample, to enhance A2aR blockade for the treatment of cancer. In someaspects, the presently disclosed subject matter involves the use ofmetabolic reprogramming agents (e.g., DON, DON prodrugs, etc.) incombination immunotherapy together with A2aR blockade, and optionally incombination with a third immunotherapy, a fourth immunotherapy, or afifth immunotherapy, such as tumor vaccines, checkpoint blockade and/oradoptive cell therapy.

In some embodiments, the method of treating cancer further includessimultaneously or sequentially administering a therapeutically effectiveamount of the second immunotherapy to the subject, wherein the secondimmunotherapy is an adenosine A2aR blockade. Exemplary A2aR inhibitorsof use in the A2aR blockade as an immunotherapy include, withoutlimitation, SCH58261, SYN115, ZM241365 and FSPTP.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with a CD73-expressingtumor, the method comprising: (a) administering a therapeuticallyeffective amount of SCH58261 to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism. Exemplary CD73-expressing tumors include, withoutlimitation, breast tumors (e.g., breast adenocarcinoma, metastaticbreast cancer) and melanoma (e.g., metastatic). In some embodiments, theCD73-expressing tumor is a metastatic tumor and administration ofSCH58261 suppresses metastases in the CD73-expressing tumor. In someembodiments, the CD73-expressing tumor is melanoma and SCH58261 and themetabolic reprogramming agent are administered in combination with ananti-PD1 antibody to prolong survival and reduce the metastatic melanomaburden. In some embodiments, the CD73-expressing tumor is breast cancerand SCH58261 and the metabolic reprogramming agent are administered incombination with an anti-PD1 antibody to prolong survival and reduce themetastatic breast cancer burden. In some embodiments, theCD73-expressing tumor is a breast cancer tumor and SCH₅₈₂₆₁ and themetabolic reprogramming agent are administered in combination with achemotherapeutic agent (e.g., doxorubicin) to increase the sensitivityof the breast cancer tumor to the chemotherapeutic agent.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with a CD73-expressingtumor, the method comprising: (a) administering a therapeuticallyeffective amount of SYN115 to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism. In some embodiments, an anti-PD-1 antibody is administeredin combination with SYN115 and the metabolic reprogramming agent.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of melanoma, the method comprising: (a)administering a therapeutically effective amount of ZM241365 to thesubject; and (b) sequentially or simultaneously administering to thesubject a therapeutically effective amount of a metabolic reprogrammingagent that decreases glutamine metabolism. In some embodiments, ananti-CTLA4 antibody is administered in combination with ZM241365 and themetabolic reprogramming agent.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of bladder cancer, the methodcomprising: (a) administering a therapeutically effective amount ofFSPTP to the subject; and (b) sequentially or simultaneouslyadministering to the subject a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism. Insome embodiments, FSPTP is administered via intratumoral injection.

4. Killer-Cell Immunoglobulin-Like Receptor (KIR) Blockade

Aspects of the presently disclosed subject matter involve the use ofmetabolic reprogramming agents (e.g., DON, DON prodrugs, e.g., compoundshaving any one of formula (I), formula (IIA), formula (IIB), or formula(III), below.) in combination immunotherapy together with killer-cellimmunoglobulin-like receptor (KIR) blockade, for example, to enhance KIRblockade for the treatment of cancer. In some aspects, the presentlydisclosed subject matter involves the use of metabolic reprogrammingagents (e.g., DON, DON prodrugs, etc.) in combination immunotherapytogether with KIR blockade, and optionally in combination with a thirdimmunotherapy, a fourth immunotherapy, and/or a fifth immunotherapy,such as tumor vaccines, adoptive cell therapy, A2aR blockade, and/orcheckpoint blockade.

In some embodiments, the method of treating cancer further includessimultaneously or sequentially administering a therapeutically effectiveamount of the second immunotherapy to the subject, wherein the secondimmunotherapy is a KIR blockade.

Exemplary KIR inhibitors of use in the KIR blockade as an immunotherapyinclude, without limitation, IPH2102/BMS-986015 (lirilumab).

In particular embodiments, the presently disclosed subject matterprovides a method of treating acute myeloid leukemia, the methodcomprising: (a) administering a therapeutically effective amount oflirilumab to the subject; and (b) sequentially or simultaneouslyadministering to the subject a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating a solid tumor, the method comprising: (a)administering a therapeutically effective amount of lirilumab to thesubject; and (b) sequentially or simultaneously administering to thesubject a therapeutically effective amount of a metabolic reprogrammingagent that decreases glutamine metabolism. In some embodiments, thesolid tumor is a melanoma tumor, and lirilumab is administered incombination with nivolumab. In some embodiments, the solid tumor is anon-small cell lung cancer tumor, and lirilumab is administered incombination with nivolumab. In some embodiments, the solid tumor is agastrointestinal tumor and lirilumab is administered in combination withnivolumab. In some embodiments, the solid tumor is a squamous cellcarcinoma of the head and neck tumor and lirilumab is administered incombination with nivolumab. In some embodiments, the solid tumor is ahepatocellular carcinoma tumor and lirilumab is administered incombination with nivolumab.

In particular embodiments, the presently disclosed subject matterprovides a method of treating a hematological tumor, the methodcomprising: (a) administering a therapeutically effective amount oflirilumab to the subject; and (b) sequentially or simultaneouslyadministering to the subject a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism. Insome embodiments, the hematological tumor is relapsed and/or refractorynon-Hodgkin's lympohoma and lirilumab is administered in combinationwith nivolumab. In some embodiments, the hematological tumor is relapsedand/or refractory Hodgkin's lympohoma and lirilumab is administered incombination with nivolumab. In some embodiments, the hematological tumoris relapsed and/or refractory multiple myeloma and lirilumab isadministered in combination with nivolumab. In some embodiments, thehematological tumor is relapsed and/or refractory chromic myelogenousleukemia and lirilumab is administered in combination with nivolumab.

In particular embodiments, the presently disclosed subject matterprovides a method of treating relapsed and/or refractory multiplemyeloma post autologous transplant, the method comprising: (a)administering a therapeutically effective amount of lirilumab to thesubject optionally in combination with elotuzumab; and (b) sequentiallyor simultaneously administering to the subject a therapeuticallyeffective amount of a metabolic reprogramming agent that decreasesglutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method of treating relapsed and/or refractory acute myeloidleukemia, the method comprising: (a) administering a therapeuticallyeffective amount of lirilumab to the subject optionally in combinationwith 5-azacytidine; and (b) sequentially or simultaneously administeringto the subject a therapeutically effective amount of a metabolicreprogramming agent that decreases glutamine metabolism.

5. Vaccines

Vaccines stimulate the body's immune system to attack abnormal ormalignant ells, such as cancer, resulting in a reduction of the thosecells. Cancer or tumor vaccines typically contain a tumor antigen in animmunogenic formulation that stimulates tumor antigen-specific helpercells, CTLs and/or B cells. Exemplary formulations of vaccines include,without limitations, dendritic cells (e.g., autologous dendritic cellspulsed with tumor cells or antigens), heterologous tumor cellstransfected with immunostimulant agents, such as GM-CSF, recombinantvirus, and/or peptides or proteins administered with adjuvants, such asCpG.

Aspects of the presently disclosed subject matter involve combinationimmunotherapy using metabolic reprogramming agents (e.g., that decreaseglutamine metabolism (e.g., DON or a DON prodrug, e.g, compounds havingany one of formula (I), formula (IIA), formula (IIB), or formula (III))sequentially or simultaneously with vaccines, for example, for thetreatment of cancer. It is believed that when used as a combinationimmunotherapy with vaccines the metabolic reprogramming agents can helpdelay or stop cancer cell growth, cause tumor shrinkage, prevent cancerfrom recurring, and/or eliminate cancer cells that have not beeneradicated by other treatments.

Accordingly, in some embodiments, the method of treating cancer furtherincludes simultaneously or sequentially administering a therapeuticallyeffective amount of the second immunotherapy to the subject, wherein thesecond immunotherapy is a vaccine (e.g., tumor vaccine).

Exemplary such vaccines include, without limitation, peptide vaccines,dendritic cell (DC) vaccines, EGFRvIII vaccines, mesothilin vaccine,G-VAX, listeria vaccines, and a dentritic cell/RCC fusion cell vaccine.

In particular embodiments, the presently disclosed subject matterprovides a method for the treatment or prevention of a humanpapillomavirus (HPV)-associated cancer in a subject in need thereof, themethod comprising: (a) administering to the subject a therapeuticallyeffective amount of a recombinant HPV vaccine; and (b) simultaneously orsequentially administering a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism.

In some embodiments, the subject has an oncogenic HPV-type 6 infectionand the rHPV vaccine comprises a rHPV type 6 vaccine. In someembodiments, the subject has an oncogenic HPV-type 11 infection and therHPV vaccine comprises a rHPV type 11 vaccine. In some embodiments, thesubject has an oncogenic HPV-type 16 infection and the rHPV vaccinecomprises a rHPV type 16 vaccine. In some embodiments, the subject hasan oncogenic HPV-type 18 infection and the rHPV vaccine comprises a rHPVtype 18 vaccine. In some embodiments, the subject is a female and thecancer is selected from the group consisting of cervical, vaginal andvulvar cancer. In some embodiments, the subject is a female between the9 and 26 years old. In some embodiments, the subject is a female between9 and 26 years old and the HPV-associated cancer is cervical cancer. Insome embodiments, the subject is a female between 9 and 26 years old andthe HPV-associated cancer is vaginal cancer. In some embodiments, thesubject is a female between 9 and 26 years old and the HPV-associatedcancer is vulvar cancer. In some embodiments, the subject is male orfemale and the HPV-associated cancer comprises anal cancer. In someembodiments, the subject is a male or female between 9 and 26 years oldand the the HPV-associated cancer is anal cancer. In some embodiments,the HPV vaccine comprises GARDASIL. In some embodiments, the subject isa female between 10 and 25 years old and the HPV-associated cancer iscervical cancer. In some embodiments, the HPV vaccine comprisesCERVARIX. In some embodiments, the HPV vaccine is administeredintramuscularly via injection as a suspension.

In particular embodiments, the presently disclosed subject matterprovides a method for the treatment or prevention of a hepatitisB-associated cancer in a subject in need thereof, the method comprising:(a) administering to the subject a therapeutically effective amount of ahepatitis B vaccine; and (b) simultaneously or sequentiallyadministering a therapeutically effective amount of a metabolicreprogramming agent that decreases glutamine metabolism. In someembodiments, the subject has a hepatitis B virus infection and thecancer is liver cancer.

6. Passive Immunotherapy

Aspects of the presently disclosed subject matter involve combinationimmunotherapy using metabolic reprogramming agents (e.g., that decreaseglutamine metabolism (e.g., DON or a DON prodrug, e.g., compounds havingany one of formula (I), formula (IIA), formula (IIB), or formula (III))sequentially or simultaneously with passive immunotherapy antibodies forthe treatment of cancer.

Accordingly, in some embodiments, the method of treating cancer furtherincludes simultaneously or sequentially administering a therapeuticallyeffective amount of the second immunotherapy to the subject, wherein thesecond immunotherapy is a passive immunotherapy antibody. As usedherein, “passive immunotherapy antibody” refers to monoclonal antibodiestargeted to cancer cell-surface specific antigens to provide cancerimmunity without actively stimulating a patient's immune system.

Exemplary passive immunotherapy antibodies of use herein include,without limitation, bevacizumab (e.g., AVASTIN), cetuximab (e.g.,ERBITUX), rituximab (e.g., RITUXAN), trastuzumab (e.g., HERCEPTIN),alemtuzumab (e.g., CAMPATH), ibritumomab tiuxetan (e.g., ZEVALIN),panitumumab (e.g., VECTIBIX).

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with metastatic colorectalcancer, the method comprising: (a) administering a therapeuticallyeffective amount of bevacizumab to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism. In some embodiments, bevacizumab is administered as a first-or second-line treatment in combination with intravenous5-fluorouracil-based chemotherapy. In some embodiments, bevacizumab isadministered as a second-line treatment in combination withfluoropyrimidine-based (combined with irinotecan or oxaliplatin)chemotherapy after cancer progression following a first-line treatmentthat includes bevacizumab.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with non-small cell lungcancer, the method comprising: (a) administering a therapeuticallyeffective amount of bevacizumab to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism. In some embodiments, the non-small cell lung cancercomprises advanced nonsquamous non-small cell lung cancer andbevacizumab is administered to subjects who have not receivedchemotherapy for their advanced disease in combination with carboplatinand paclitaxel.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with plantinum-resistantovarian cancer, the method comprising: (a) administering atherapeutically effective amount of bevacizumab to the subject; and (b)sequentially or simultaneously administering to the subject atherapeutically effective amount of a metabolic reprogramming agent thatdecreases glutamine metabolism. In some embodiments, the subject has hadno more than two prior chemotherapy treatments and bevacizumab is usedto treat plantinum-resistant recurrent epithelial ovarian, fallopiantube or primary peritoneal cancer in the subject in combination withpaclitaxel, pegylated liposomal doxorubicin or topotecan.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with advanced cervicalcancer, the method comprising: (a) administering a therapeuticallyeffective amount of bevacizumab to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism. In some embodiments, the subject has persistent, recurrent,or metastatic cervical cancer and bevacizumab is administered incombination with paclitaxel and cisplatin. In some embodiments, thesubject has persistent, recurrent, or metastatic cervical cancer andbevacizumab is administered in combination with paclitaxel andtopotecan.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with metastatic renal cellcarcinoma, the method comprising: (a) administering a therapeuticallyeffective amount of bevacizumab to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism. In some embodiments, the subject has metastatic kidneycancer and bevacizumab is administered in combination with interferonalfa.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with recurrentglioblastoma, the method comprising: (a) administering a therapeuticallyeffective amount of bevacizumab to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism. In some embodiments, the subject with recurrent glioblastomais an adult.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with head and neck cancer,the method comprising: (a) administering a therapeutically effectiveamount of cetuximab to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism. In some embodiments, the subject has locally or regionallyadvanced squamous cell carcinoma of the head and neck and cetuximab isadministered in combination with radiotherapy. In some embodiments, thesubject has recurrent locoregional disease or metastatic squamous cellcarcinoma of the head and neck and cetuximab is administered incombination with platinum-based therapy with 5-FU. In some embodiments,the subject has recurrent or metastatic squamous cell carcinoma of thehead and neck and has failed to respond to prior platinum-based therapy.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with metastatic colorectalcancer, the method comprising: (a) administering a therapeuticallyeffective amount of cetuximab to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism. In some embodiments, the metastatic colorectal cancercomprises KRAS wild-type, epidermal growth factor rector(EGFR)-expressing, metastatic colorectal cancer, and cetuximab isadministered in combination with FOLFIRI (irinotecan, 5-fluorouracil,and lucovorin). In some embodiments, the metastatic colorectal cancercomprises KRAS wild-type, epidermal growth factor rector(EGFR)-expressing, metastatic colorectal cancer, the subject isrefractory to irinotecan-based chemotherapy, and cetuximab isadministered in combination with irinotecan. In some embodiments, themetastatic colorectal cancer comprises KRAS wild-type, epidermal growthfactor rector (EGFR)-expressing, metastatic colorectal cancer, and thesubject has failed to respond to oxaliplatin and irinotecan-basedchemotherapy. In some embodiments, the metastatic colorectal cancercomprises KRAS wild-type, epidermal growth factor rector(EGFR)-expressing, metastatic colorectal cancer, and the subject isinteolerant to irinotecan.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with non-Hodkin's lymphoma,the method comprising: (a) administering a therapeutically effectiveamount of rituximab to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism.

In some embodiments, the subject has recurrent or refractory low-gradeor follicular CD20-positive non-Hodgkin's lymphoma. In some embodiment,the subject has newly diagnosed CD20-positive non-Hodgkin's lymphoma. Insome embodiments, the subject has low-grade or follicular CD20-positivenon-Hodgkin's lymphoma and responded to initial treatment with CVPchemotherapy (cyclophosphamide, vincristine and prednisone). In someembodiments, the subject has CD20-positive diffuse large B-cellnon-Hodgkin's lymphoma and the rituximab is administered in combinationwith CHOP chemotherapy (cyclophosphamide, doxorubicin hydrochloride,vincristine and prednisolone). In some embodiments, the subject hasnewly disagnosed or recurrent CD20-positive chronic lymphocytic leukemiaand the rituximab is administered in combination with FC chemotherapy(fludarabine and cyclophosphamide).

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with early-stage breastcancer that is human epidermal growth factor receptor 2-positive(HER2+), the method comprising: (a) administering a therapeuticallyeffective amount of alemtuzumab to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism.

In some embodiments, alemtuzumab is administered to the subject as partof a treatment course comprising doxorubicin, cyclophosphamide andeither paclitaxel or docetaxel. In some embodiments, alemtuzumab isadministered to the subject with docetaxel and carboplatin. In someembodiments, alemtuxumab is administered to the subject after treatmentwith anhtracylcine-based thereapy. In some embodiments, the HER2+ breastcancer has spread into the subject's lymph nodes. In some embodiments,the HER2+ breast cancer has not spread into the subject's lymph nodesand the cancer is estrogen receptor/progesterone receptor(ER/PR)-negative or has one high risk feature selected from the groupconsisting of a tumor size >2 cm, age <35 years, or tumor grade of 2 or3.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with B-cell chroniclymphocytic leukemia (B-CLL), the method comprising: (a) administering atherapeutically effective amount of alemtuzumab to the subject; and (b)sequentially or simultaneously administering to the subject atherapeutically effective amount of a metabolic reprogramming agent thatdecreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with low-grade orfollicular B-cell non-Hodgkin's lymphoma (NHL) that has relapsed duringor after treatment with other anticancer drugs or newly diagnosedfollicular NHL following a response to initial anticancer therapy, themethod comprising: (a) administering a therapeutically effective amountof ibritumomab tiuxetan to the subject; and (b) sequentially orsimultaneously administering to the subject a therapeutically effectiveamount of a metabolic reprogramming agent that decreases glutaminemetabolism. In some embodiments, administering ibritumomab tiuxetancomprises intravenous injection comprising two infusions of rituximab(e.g., to reduce the number of B-cells in the subject's blood) and oneinjection of Yttrium-90 ibritumomab tiuxetan.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with wild-type KRAS (exon 2in codons 12 or 13) metastatic colorectal cancer, the method comprising:(a) administering a therapeutically effective amount of panitumumab tothe subject as a first-line therapy in combination with folinic acid,fluorouracil and oxaliplatin; and (b) sequentially or simultaneouslyadministering to the subject a therapeutically effective amount of ametabolic reprogramming agent that decreases glutamine metabolism.

In particular embodiments, the presently disclosed subject matterprovides a method for treatment of a subject with wild-type KRAS (exon 2in codons 12 or 13) metastatic colorectal cancer, the method comprising:(a) administering a therapeutically effective amount of panitumumabfollowing disease progression after prior treatment withfluoropyrimidine, oxalipltin, and irinotecan-containing chemotherapy;and (b) sequentially or simultaneously administering a therapeuticallyeffective amount of a metabolic reprogramming agent that decreasesglutamine metabolism.

7. Combination Immunotherapy

Aspects of the presently disclosed subject matter involve the use ofmetabolic reprogramming agents (e.g., DON, DON-prodrugs, e.g., compoundshaving any one of formula (I), formula (IIA), formula (IIB), or formula(III)) in combination immunotherapy together with a second, third,fourth, and/or fifth immunotherapy, for example, to enhance theimmunotherapy for the treatment of cancer.

In some embodiments, the method of treating cancer further includessimultaneously or sequentially administering a therapeutically effectiveamount of a third immunotherapy to the subject, wherein the thirdimmunotherapy is selected from the group consisting of checkpointblockade, adoptive cell therapy, CAR-T cell therapy, marrow-infiltratinglymphocytes, A2aR blockade, KIR blockade, vaccines (e.g., tumorvaccines), passive immunotherapy antibodies, and combinations thereof.

In some embodiments, the method of treating cancer further includessimultaneously or sequentially administering a therapeutically effectiveamount of a third and/or fourth immunotherapy to the subject, whereinthe third and/or fourth immunotherapy is selected from the groupconsisting of checkpoint blockade, adoptive cell therapy, CAR-T celltherapy, marrow-infiltrating lymphocytes, A2aR blockade, KIR blockade,vaccines (e.g., tumor vaccines), passive immunotherapy antibodies, andcombinations thereof.

In some embodiments, the method of treating cancer further includessimultaneously or sequentially administering a therapeutically effectiveamount of a third, fourth, and/or fifth immunotherapy to the subject,wherein the third, fourth, and/or fifth immunotherapy is selected fromthe group consisting of checkpoint blockade, adoptive cell therapy,CAR-T cell therapy, marrow-infiltrating lymphocytes, A2aR blockade, KIRblockade, vaccines (e.g., tumor vaccines), passive immunotherapyantibodies, and combinations thereof

8. Adjuvant to Cancer Therapy

Aspects of the presently disclosed subject matter involve the use ofmetabolic reprogramming agents (e.g., DON or a DON prodrug, e.g.,compounds having any one of formula (I), formula (IIA), formula (IIB),or formula (III)) as an adjuvant to cancer therapy, for example, for thetreatment or prevention of cancer.

Accordingly, in some embodiments, the method of treating cancer furtherincludes simultaneously or sequentially administering to the subject atherapeutically effective amount of a cancer therapy selected from thegroup consisting of: (i) chemotherapy; (ii) photodynamic therapy; (iii)proton therapy; (iv) radiotherapy; (v) surgery; and combinationsthereof.

In some embodiments, the first immunotherapy, and the secondimmunotherapy if administered, is/are administered to the subject in theabsence of chemotherapy. In some embodiments, the first immunotherapy,and the second immunotherapy if administered, is/are administered to thesubject in the absence of photodynamic therapy. In some embodiments, thefirst immunotherapy, and the second immunotherapy if administered,is/are administered to the subject in the absence of proton therapy. Insome embodiments, the first immunotherapy, and the second immunotherapyif administered, is/are administered to the subject in the absence ofradiotherapy. In some embodiments, the first immunotherapy, and thesecond immunotherapy if administered, is/are administered to the subjectin the absence of surgery.

II. Metabolic Reprogramming Agents

The presently disclosed subject matter contemplates the use of variousagents in connection with the methods, uses, and compositions describedherein. Certain of the methods and compositions described herein relateto the metabolic reprogramming of cells using at least one metabolicreprogramming agent described herein to treat conditions, diseases, ordisorders that involve metabolically reprogrammed cells whoseactivation, function, growth, proliferation, and/or survival depends onincreased activity of at least one, at least two, or at least threemetabolic pathways selected from the group consisting of glutaminemetabolism, glycolysis, and fatty acid synthesis. Aspects of the methodsand compositions described herein relate to the use of least onemetabolic reprogramming agent described herein to treat conditions,diseases, or disorders that involve aberrant and/or excessive amounts ofat least one, at least two, or at least three metabolic pathwaysselected from the group consisting of aberrant and/or excessiveglutamine metabolism, aberrant and/or excessive glycolysis, or aberrantand/or excessive fatty acid synthesis.

As used herein, “metabolic reprogramming agent” generally refers to anagent that modulates the metabolic activity of at least one metabolicpathway in a cell, for example, to alter activation, function, growth,proliferation, and/or survival of the cell. As used herein, “modulate”broadly means to cause or facilitate a qualitative or quantitativechange, alteration, or modification in a molecule, a process, pathway,or phenomenon of interest. Without limitation, such change may be anincrease, decrease, a change in binding characteristics, or change inrelative strength or activity of different components or branches of theprocess, pathway, or phenomenon. The term “modulator” broadly refers toany molecule or agent that causes or facilitates a qualitative orquantitative change, alteration, or modification in a process, pathway,or phenomenon of interest. As used herein, the term “modulator”comprises both inhibitors and activators of a metabolic pathway ortarget. For example, “modulator” comprises both inhibitors andactivators of expression and/or activity of a component involvedglutamine metabolism, a component involved in glycolysis, and/or acomponent involved in fatty acid metabolism (e.g., fatty acid synthesisor fatty acid oxidation).

As used herein, the phrase “modulation of a metabolic pathway” refers tomodulation of activity of at least one component of the metabolicpathway. It is contemplated herein that modulator of the metabolicpathway can be, for example, a receptor ligand (e.g., a small molecule,an antibody, a siRNA), a ligand sequestrant (e.g., an antibody, abinding protein), a modulator of phosphorylation of a pathway componentor a combination of such modulators. One of skill in the art can easilytest an agent to determine if it modulates a metabolic pathway byassessing, for example, phosphorylation status of a receptor orexpression or synthesis of downstream proteins or enzymes controlled bythe pathway in cultured cells and comparing the results to cells nottreated with a modulator. A modulator is determined to be a metabolicpathway modulator if the level of phosphorylation of the receptor orexpression of or synthesis of downstream proteins or enzymes in aculture of cells is reduced by at least 20% compared to the level ofphosphorylation of the receptor or expression or synthesis of downstreamproteins or enzymes in cells that are cultured in the absence of themodulator; preferably the level of phosphorylation or expression orsynthesis of downstream proteins or enzymes is altered by at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 99% in the presence of a metabolicpathway modulator.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction”, “decrease” or “inhibit” means a decrease by at least 10% ascompared to a reference level, for example a decrease by at least about20%, or at least about 30%, or at least about 40%, or at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90%, where the decrease is less than 100%. In oneembodiment, the decrease includes a 100% decrease (e.g. absent level ascompared to a reference sample), or any decrease between 10-100% ascompared to a reference level.

The terms “increased”, ‘increase”, “enhance” or “activate” are all usedherein to generally mean an increase by a statically significant amount;for the avoidance of any doubt, the terms “increased”, “increase”,“enhance” or “activate” means an increase of at least 10% as compared toa reference level, for example an increase of at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% increase or any increasebetween 10-100% as compared to a reference level, or at least about a2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level.

Certain methods, compositions, and agents contemplated herein modulatean immune response. In the contexts of decreasing an immune response(e.g., inhibiting an immunosuppressive pathway, e.g., via checkpointblockade, A2aR blockade, KIR blockade, etc.), the methods, compositions,and agents contemplated herein can decrease the immune response by atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80, 90%, or as much as100% as compared to a reference level (e.g., an objective measure of theimmune response before employing the method, composition, and/or agent).In the contexts of increasing an immune response (e.g., activating aT-cell co-stimulatory signal), the methods, compositions, and agentscontemplated herein can increase the immune response by at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80, 90%, or as much as 100%, at leastabout a 2-fold, or at least about a 3-fold, or at least about a 4-fold,or at least about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level (e.g., an objective measure of the immune responsebefore employing the method, composition, and/or agent).

Certain methods, compositions, and agents contemplated herein modulategrowth, proliferation, metastasis, and invasion of malignant cells(e.g., cancer cells). In the contexts of inhibiting growth,proliferation, metastasis, invasion, and/or survival of malignant cells(e.g., cancer cells), the methods, compositions, and agents contemplatedherein can decrease the growth, proliferation, metastasis, invasion,and/or survival of malignant cells (e.g., cancer cells) by at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80, 90%, or as much as 100% ascompared to a reference level (e.g., an objective measure of the growth,proliferation, metastasis, invasion, and/or survival of malignant cellsbefore employing the method, composition, and/or agent).

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value.

As used more particularly herein in some contexts, “modulates”,“modulating”, and “modulation” are used interchangeably and refer to anyone or a combination of a decrease in glutamine metabolism, a decreasein glycolysis, and a decrease in fatty acid synthesis. In othercontexts, “modulates”, “modulating”, and “modulation” are usedinterchangeably and refer to any one or a combination of an increase inglutamine metabolism, an increase in glycolysis, and an increase infatty acid synthesis. In certain contexts, “modulates”, “modulating”,and “modulation” are used interchangeably and refer to any one or acombination of an increase in oxidative phosphorylation.

Glutamine (2-amino-4-carbamoylbutanoic acid), is used by the cell forboth bioenergetic and biosynthetic needs. Glutamine can be used as anamino acid for protein synthesis, as a carbon source, or as the primarynitrogen donor for multiple essential biosynthetic reactions in thecell. Once taken up by the cell, much of the glutamine is converted toglutamate by mitochondrial glutaminase. Both glutamine and glutamatecontribute to anabolic metabolism; glutamine supplies nitrogen fornucleotide and hexosamine synthesis while glutamate is the nitrogendonor for the synthesis of many nonessential amino acids. Glutamate canbe used to support the production of NADPH or converted to the metabolicintermediates pyruvate and α-ketoglutarate. As used herein, the term“glutamine metabolism” or “glutamine metabolic activity” refers to thechemical reactions, enzymes, and pathways involving glutamine. As usedherein, the term “glutamine metabolic pathway” is a biochemical pathwaythat involves glutamine.

As can be envisioned by a person with skill in the art, a metabolicreprogramming agent can modulate any of the chemical reactions, enzymesand/or pathways involving glutamine. In some embodiments, at least onemetabolic reprogramming agent can modulate chemical reactions, enzymesand/or pathways that do not directly involve glutamine, such as theconversion of pyruvate to acetyl CoA or the citric acid cycle, butindirectly affect any of the chemical reactions, enzymes and/or pathwaysinvolving glutamine. Certain methods, compositions, and metabolicreprogramming agents contemplated herein decrease glutamine metabolismin cells. In the context of decreasing glutamine metabolism in cells,the methods, compositions, and agents contemplated herein can decreaseglutamine metabolism in cells by at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80, 90%, or as much as 100% as compared to a reference level(e.g., an objective measure of the glutamine metabolic activity beforeemploying the method, composition, and/or agent).

In some embodiments, at least one metabolic reprogramming agent is aglutamine antagonist (i.e., an agent that decrease glutaminemetabolism). As used herein, the term “glutamine antagonist” refers toan agent that blocks or interferes with the synthesis or use ofglutamine in a cell, and preferably in a cell that is part of a livingorganism. When it is said that the glutamine antagonist interferes withthe synthesis of glutamine, it is meant that the antagonist acts toreduce the amount or rate of glutamine synthesis to less than the amountor rate that would be experienced in the absence of the glutamineantagonist. When it is said that the glutamine antagonist interfereswith the use of glutamine, it is meant that the antagonist acts toinhibit or block a metabolic pathway downstream of glutamine, that is, apathway in which glutamine acts as a precursor of one or morenon-glutamine compounds, or that the antagonist acts to depleteglutamine in a cell or an organism by reacting the glutamine to form anon-glutamine product, or by reversibly or irreversibly binding withglutamine to reduce its availability.

In some embodiments, at least one metabolic reprogramming agent of thepresently disclosed subject matter can be a glutamine analog thatinterferes with a glutamine metabolic pathway, an antagonist thatinhibits the synthesis of glutamine, a glutamine depleting enzyme, acompound that reacts with glutamine under intracellular conditions toform a non-glutamine product, an antagonist that inhibits glutamineuptake by cells, an agent that inhibits glutamine oxidation, an agentthat inhibits a glutamine transporter, an agent that inhibitsglutaminolysis (a series of biochemical reactions by which glutamine islysed to glutamate, aspartate, carbon dioxide, pyruvate, lactate,alanine and/or citrate), or a glutamine binding compound that reducesthe biological availability of glutamine. It should be recognized that acompound that is a useful metabolic reprogramming agent may have two ormore of these characteristics. For example, a compound that is aglutamine analog that interferes with a glutamine metabolic pathwaymight also act as an antagonist that inhibits the synthesis ofglutamine.

In some embodiments, at least one metabolic reprogramming agent can bean antagonist that inhibits the synthesis of glutamine. Examples ofcompounds having this activity include inhibitors of glutamine synthase(EC 6.3.1.2), such as L-methionine-DL-sulfoximine, and phosphinothricin;inhibitors of glutamate synthase (EC 1.4.1.13); inhibitors ofamidophosphoribosyltransferase (EC 2.4.2.14); and inhibitors ofglutamate dehydrogenase; and mixtures of any two or more of these.

In some embodiments, at least one metabolic reprogramming agent can be aglutamine depleting enzyme. Examples of such enzymes includecarbamoyl-phosphate synthase (EC 6.3.5.5), glutamine-pyruvatetransaminase (EC 2.6.1.15), glutamine-tRNA ligase (EC 6.1.1.18),glutaminase (EC 3.5.1.2), D-glutaminase (EC 3.5.1.35), glutamineN-acyltransferase (EC2.3.1.68), glutaminase-asparaginase (in particularglutaminase-asparaginase of Pseudomonas 7a and Acinatobacter sp.), andmixtures of any two or more of these.

In some embodiments, at least one metabolic reprogramming agent can be acompound that reacts with glutamine under intracellular conditions toform a non-glutamine product. An example of a compound having thisproperty is phenylbutyrate (See Darmaun et al., Phenylbutyrate-induceglutamine depletion in humans: effect on leucine metabolism, pp.E801-E807, in Glutamine Depletion and Protein Catabolism, Am. Physiol.Soc. (1998)). Another example of a glutamine antagonist having thischaracteristic is phenylacetate (See, U.S. Pat. No. 6,362,226).

In some embodiments, at least one metabolic reprogramming agent can bean antagonist that inhibits glutamine uptake by cells. Examples ofcompounds having this property include alpha-methylaminoisobutyric acid(inhibits GynT plasma membrane glutamine transporter; See, Varoqui etal., J. Biol. Chem., 275(6):4049-4054 (2000), wortmannin, and LY-294002(inhibits hepatic glutamine transporter; See, Pawlik et al., Am. J.Physiol. Gastrointest. Liver Physiol., 278:G532-G541 (2000)).

In some embodiments, at least one metabolic reprogramming agent can be aglutamine binding compound that reduces the biological availability ofglutamine.

In some embodiments, at least one metabolic reprogramming agent can be aglutamine analog that interferes with a glutamine metabolic pathway(e.g., decreases glutamine metabolism/metabolic activity). Examples ofcompounds that can act in this manner include acivicin (L-(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), DON(6-diazo-5-oxo-L-norleucine), azaserine, azotomycin, chloroketone(L-2-amino-4-oxo-5-chloropentanoic acid),N³-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) (inactivatesglucosamine-6-phosphate synthase (EC 2.6.1.16), See, Zgódka et al.,Microbiology, 147:1955-1959 (2001)), (3S,4R)-3,4-dimethyl-L-glutamine,(3S,4R)-3,4-dimethyl-L-pyroglutamic acid (See, Acevedo et al.,Tetrahedron., 57:6353-6359 (2001)),1,5-N,N′-disubstituted-2-(substituted benzenesulphonyl) glutamamides(See, Srikanth et al., Bioorganic and Medicinal Chemistry, (2002)), or amixture of any two or more of these. In some embodiments, at least onemetabolic reprogramming agent is selected from the group consisting ofacivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), azaserine,6-diazo-5-oxo-norleucine (DON), and 5-diazo-4-oxo-L-norvaline (L-DONV).

In some embodiments, at least one metabolic reprogramming agent is aprodrug of a glutamine analog that interferes with a glutamine metabolicpathway (e.g., decreases glutamine metabolism/metabolic activity). Insome embodiments, at least one metabolic reprogramming agent is aprodrug of acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), azaserine,6-diazo-5-oxo-norleucine (DON), and 5-diazo-4-oxo-L-norvaline (L-DONV).Suitable exemplary prodrugs of acivicin, azaserine, DON, and L-DONV canbe found in “Prodrugs of Glutamine Analogs” (PCT/US2016/044767 (WO2017/023774 A1), and herein incorporated by reference in its entirety).

Glycolysis is the metabolic pathway that converts glucose into pyruvatewith the concurrent production of ATP. Pyruvate is a metabolicintermediate that can then enter the tricarboxylic acid (TCA) cyclewithin mitochondria to produce NADH and FADH₂. The first step inglycolysis is the phosphorylation of glucose by hexokinase to formglucose 6-phosphate.

In some embodiments, at least one metabolic reprogramming agent canmodulate any of the chemical reactions and/or enzymes involved inglycolysis. In some embodiments, at least one metabolic reprogrammingagent can modulate chemical reactions, enzymes and/or pathways that donot directly involve glycolysis, but indirectly affect any of thechemical reactions, enzymes and/or pathways involving glycolysis.Certain methods, compositions, and metabolic reprogramming agentscontemplated herein decrease glycolysis in cells. In the context ofdecreasing glycolysis in cells, the methods, compositions, and agentscontemplated herein can decrease glycolysis in cells by at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80, 90%, or as much as 100% ascompared to a reference level (e.g., an objective measure of theglycolytic metabolic activity before employing the method, composition,and/or agent). As used herein, the term “glycolytic metabolic activity”refers to the chemical reactions and enzymes involving the glycolysispathway.

In some embodiments, at least one metabolic reprogramming agent of thepresently disclosed subject matter can be an agent that interferes withglycolysis or a related pathway that affects glycolysis; an agent thatinhibits the synthesis of pyruvate and/or one of the intermediateproducts of glycolysis; an agent that inhibits one or more of theenzymes involved in glycolysis, such as hexokinase, phosphoglucoseisomerase, phosphofructokinase, fructose-bisphosphate aldolase,triophosphate isomerase, glyceraldehyde phosphate dehydrogenase,phosphoglycerate kinase, phosphoglycerate mutase, enolase, and/orpyruvate kinase; an agent that depletes glucose-6-phosphate, one of therate-limiting products in glycolysis; an agent that inhibits glucoseuptake and/or transport across the plasma membrane by cells; or aglucose binding compound that reduces the biological availability ofglucose. It should be recognized that a compound that is a usefulmetabolic reprogramming agent may have two or more of thesecharacteristics.

In some embodiments, at least one metabolic reprogramming agentinterferes or inhibits the expression and/or activity of hexokinase.Examples of inhibitors of hexokinase include, but are not limited to,2-deoxyglucose (2-DG), 3-bromopyruvate (3-BrPA), lonidamine (LND),sodium fluoride, and potassium fluoride. In some embodiments, at leastone metabolic reprogramming agent is 2-deoxy-D-glucose (2-DG).

Fatty acid synthesis is the process in the cell that creates fatty acidsfrom acetyl-CoA and malonyl-CoA precursors. Fatty acid oxidation is theprocess by which fatty acid molecules are broken down in themitochondria to generate acetyl-CoA, which enters the citric acid cycle,and NADH and FADH₂, which are used in the electron transport chain. Theenzyme AMP-activated protein kinase (AMPK) plays a role in cellularenergy homeostasis and is a stimulator of fatty acid oxidation.

In some embodiments, at least one metabolic reprogramming agent canmodulate any of the chemical reactions and/or enzymes involved in fattyacid synthesis and/or fatty acid oxidation. In some embodiments, atleast one metabolic reprogramming agent can modulate chemical reactions,enzymes and/or pathways that do not directly involve fatty acidsynthesis and/or fatty acid oxidation, but indirectly affect any of thechemical reactions, enzymes and/or pathways involving fatty acidsynthesis and/or fatty acid oxidation.

Certain methods, compositions, and metabolic reprogramming agentscontemplated herein decrease fatty acid synthesis and/or increase fattyacid oxidation in cells. In the context of decreasing fatty acidsynthesis and/or increasing fatty acid oxidation in cells, the methods,compositions, and agents contemplated herein can decrease fatty acidsynthesis and/or increase fatty acid oxidation in cells by at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80, 90%, or as much as 100% ascompared to a reference level (e.g., an objective measure of thesynthesis of fatty before employing the method, composition, and/oragent).

In some embodiments, at least one metabolic reprogramming agent of thepresently disclosed subject matter can be an agent that interferes withfatty acid synthesis and/or fatty acid oxidation or a related pathwaythat affects fatty acid synthesis and/or fatty acid oxidation; an agentthat increases fatty acid oxidation; an agent that increases one or moreof the products of fatty acid oxidation; an agent that increases theexpression and/or activity of one or more of the enzymes involved infatty acid oxidation, such as acyl CoA dehydrogenase, enoyl CoAhydratase, 3-hydroxyacyl-CoA dehydrogenase, and β-ketothiolase; an agentthat increases expression and/or activity of AMP-activated proteinkinase (AMPK); an agent that increases uptake and/or transfer ofactivated fatty acids across the mitochondrial membrane; and an agentthat increases the expression and/or activity of enzymes involved in theuptake and/or transfer of activated fatty acids across the mitochondrialmembrane. It should be recognized that a compound that is a usefulmetabolic reprogramming agent may have two or more of thesecharacteristics. In some embodiments, at least one metabolicreprogramming agent is an activator of 5′ AMP-activated protein kinase(AMPK) activity.

At least one metabolic reprogramming agent that is an activator of AMPKactivity can be an agent that increases concentrations of AMP in thecell; an AMP analogue, such as 5-amino-4-imidazolecarboxamide ribotide(ZMP); an agent that increases phosphorylation of AMPK, such as an agentthat increases the expression and/or activity of a kinase that canphosphorylate AMPK; and an agent that is an allosteric modulator ofAMPK, such as one that can modify AMPK to make it a better substrate fora kinase that can phosphorylate AMPK.

In some embodiments, at least one metabolic reprogramming agent ismetformin.

It should be appreciated that modulation of glutamine metabolism,glycolysis, and fatty acid metabolism may result in modulation of one ormore genes or expression products of genes or biosynthesis ordegradation of one or more enzymes.

The term “expression” means the process by which information from a geneor nucleic acid (e.g., DNA) is used in the synthesis of gene products(e.g., mRNA, RNA and/or proteins) and includes, but is not limited to,one or more of the steps of replication, transcription and translation.The steps of expression which may be modulated by the agentscontemplated herein may include, for example, transcription, splicing,translation and post-translational modification of a protein. Thoseskilled in the art will appreciate that the method of modulating anyparticular protein may depend on the type of protein (e.g., proteinkinase, transcriptional regulator, enzyme, etc.), its function (e.g.,transcriptional regulation, catalysis, phosphorylation, signaltransduction, etc.), and its subcellular localization (e.g.,extracellular space, cytoplasm, nucleus, membrane, etc.). Those skilledin the art will readily appreciate appropriate agents to be used formodulation depending on the particular context (e.g., type of protein,biological function, subcellular localization, composition, method ofuse, mode of inhibition, etc.). For example, an agent can be used toinhibit enzymatic activity of an enzyme (e.g., at least one metabolicreprogramming agent that inhibits glutaminolysis catalyzed byglutaminase (e.g., a glutamine antagonist), at least one metabolicreprogramming agent that inhibits glycolysis catalyzed in part byhexokinase (e.g., 2-DG), etc.), inhibits the level or activity ofphosphorylation of a protein kinase, inhibit activation of transcriptionor a signaling pathway.

The metabolic reprogramming agents, chemotherapeutic agents, cytotoxicagents, immunotherapeutic agents, immunosuppressant agents,radiotherapeutic agents, anti-inflammatory agents, and neuroprotectiveagents described herein can be any type of agent. Exemplary types ofagents that can be used as such agents in the methods, compositions, anduses described herein include small organic or inorganic molecules;saccharides; oligosaccharides; polysaccharides; a biologicalmacromolecule selected from the group consisting of peptides, proteins,peptide analogs and derivatives; peptidomimetics; nucleic acids selectedfrom the group consisting of siRNAs, shRNAs, antisense RNAs, ribozymes,dendrimers and aptamers; an extract made from biological materialsselected from the group consisting of bacteria, plants, fungi, animalcells, and animal tissues; naturally occurring or syntheticcompositions; microcarrier or nanocarrier consisting of one or morepolymers, proteins, nucleic acids, lips, or metals; and any combinationthereof.

As used herein, the term “small molecule” can refer to agents that are“natural product-like,” however, the term “small molecule” is notlimited to “natural product-like” agents. Rather, a small molecule istypically characterized in that it contains several carbon-carbon bonds,and has a molecular weight of less than 5000 Daltons (5 kD), preferablyless than 3 kD, still more preferably less than 2 kD, and mostpreferably less than 1 kD. In some cases it is preferred that a smallmolecule have a molecular weight equal to or less than 700 Daltons.

As used herein, an “RNA interference molecule” refers to an agent whichinterferes with or inhibits expression of a target gene or genomicsequence by RNA interference (RNAi). Such RNA interfering agentsinclude, but are not limited to, nucleic acid molecules including RNAmolecules which are homologous to the target gene or genomic sequence,or a fragment thereof, short interfering RNA (siRNA), short hairpin orsmall hairpin RNA (shRNA), microRNA (miRNA) and small molecules whichinterfere with or inhibit expression of a target gene by RNAinterference (RNAi).

The term “polynucleotide” is used herein interchangeably with “nucleicacid” to indicate a polymer of nucleosides. Typically a polynucleotideis composed of nucleosides that are naturally found in DNA or RNA (e.g.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine,

deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds.However the term encompasses molecules comprising nucleosides ornucleoside analogs containing chemically or biologically modified bases,modified backbones, etc., whether or not found in naturally occurringnucleic acids, and such molecules may be preferred for certainapplications. Where this application refers to a polynucleotide it isunderstood that both DNA, RNA, and in each case both single- anddouble-stranded forms (and complements of each single-stranded molecule)are provided. Polynucleotide sequence” as used herein can refer to thepolynucleotide material itself and/or to the sequence information (e.g.the succession of letters used as abbreviations for bases) thatbiochemically characterizes a specific nucleic acid. A polynucleotidesequence presented herein is presented in a 5′ to 3′ direction unlessotherwise indicated.

The nucleic acid molecules that modulate the metabolic pathways ortargets described herein can, in some embodiments, be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. Proc. Natl. Acad. Sci. USA 91:3054-3057, 1994). The pharmaceutical preparation of the gene therapyvector can include the gene therapy vector in an acceptable diluent, orcan comprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The terms “polypeptide” as used herein refers to a polymer of aminoacids. The terms “protein” and “polypeptide” are used interchangeablyherein. A peptide is a relatively short polypeptide, typically betweenabout 2 and 60 amino acids in length. Polypeptides used herein typicallycontain amino acids, such as the 20 L-amino acids that are most commonlyfound in proteins. However, other amino acids and/or amino acid analogsknown in the art can be used. One or more of the amino acids in apolypeptide may be modified, for example, by the addition of a chemicalentity, such as a carbohydrate group, a phosphate group, a fatty acidgroup, a linker for conjugation, functionalization, etc. A polypeptidethat has a non-polypeptide moiety covalently or non-covalentlyassociated therewith is still considered a “polypeptide”. Exemplarymodifications include glycosylation and palmitoylation. Polypeptides maybe purified from natural sources, produced using recombinant DNAtechnology, synthesized through chemical means, such as conventionalsolid phase peptide synthesis, etc. The term “polypeptide sequence” or“amino acid sequence” as used herein can refer to the polypeptidematerial itself and/or to the sequence information (e.g., the successionof letters or three letter codes used as abbreviations for amino acidnames) that biochemically characterizes a polypeptide. A polypeptidesequence presented herein is presented in an N-terminal to C-terminaldirection unless otherwise indicated.

The term “identity” as used herein refers to the extent to which thesequence of two or more nucleic acids or polypeptides is the same. Thepercent identity between a sequence of interest and a second sequenceover a window of evaluation, e.g., over the length of the sequence ofinterest, may be computed by aligning the sequences, determining thenumber of residues (nucleotides or amino acids) within the window ofevaluation that are opposite an identical residue allowing theintroduction of gaps to maximize identity, dividing by the total numberof residues of the sequence of interest or the second sequence(whichever is greater) that fall within the window, and multiplying by100. When computing the number of identical residues needed to achieve aparticular percent identity, fractions are to be rounded to the nearestwhole number. Percent identity can be calculated with the use of avariety of computer programs known in the art. For example, computerprograms, such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generatealignments and provide percent identity between sequences of interest.The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl.Acad. Sci. USA 87:22264-2268, 1990) modified as in Karlin and Altschul,Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993 is incorporated into theNBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J. MoTBiol. 215:403-410, 1990). To obtain gapped alignments for comparisonpurposes, Gapped BLAST is utilized as described in Altschul et al.(Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997). Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs may be used. A PAM250 or BLOSUM62 matrix may beused. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (NCBI). Seethe Web site having URL www.ncbi.nlm.nih.gov for these programs. In aspecific embodiment, percent identity is calculated using BLAST2 withdefault parameters as provided by the NCBI.

Generally, at least one metabolic reprogramming agent described hereincan be used in combination with an additional therapeutic agent (e.g., apharmaceutically active agent, e.g., a drug approved by a regulatoryagency). The therapeutic agent may act synergistically with the agentdescribed herein, or they may independently exert their intendedeffects. The disclosure contemplates any therapeutic agent which askilled artisan would use in connection with a method, use, orcomposition described herein. Examples of therapeutic agentscontemplated for use in the presently disclosed methods, uses andcompositions in combination with the metabolic reprogramming agentsinclude, but are not limited to, chemotherapeutic agents/chemotherapy,immunotherapeutic agents/immunotherapy, immunosuppressant agents,anti-inflammatory agents, neuroprotective agents, neuroregenerativeagents, neurotrophic factors, radiotherapeutic agents/radiotherapy,proton therapy, photodynamic therapy, and stem and progenitor cells usedto replace and/or repair endogenous populations of abnormal, harmful, orunhealthy cells.

Chemotherapy and chemotherapeutic agnet are used synonymously herein. A“chemotherapeutic agent” is used to connote a compound or compositionthat is administered in the treatment of cancer. Chemotherapeutic agentscontemplated for use in combination with at least one metabolicreprogramming agent, at least two metabolic reprogramming agents, or atleast three metabolic reprogramming agents described herein include, butare not limited to, alkylating agents, such as thiotepa andcyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan andpiposulfan; aziridines, such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards, such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas, such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics, such as aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin,detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid,nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites, such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues, such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens, such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals,such as aminoglutethimide, mitotane, trilostane; folic acidreplenishers, such as folinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene;edatraxate; defofamine; demecolcine; diaziquone; elformithine;elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine;pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, e.g., paclitaxel and docetaxel;chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinumanalogs, such as cisplatin and carboplatin; vinblastine; platinum;etoposide; ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine; retinoic acid; esperamicins; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Chemotherapeutic agents also include anti-hormonal agents thatact to regulate or inhibit hormone action on tumors, such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgens,such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

In some embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D,etoposide, topotecan HCl, teniposide, and irinotecan, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese.

In some embodiments, the chemotherapeutic agent is an anti-metabolite.An anti-metabolite is a chemical with a structure that is similar to ametabolite required for normal biochemical reactions, yet differentenough to interfere with one or more normal functions of cells, such ascell division. Anti-metabolites include, but are not limited to,gemcitabine, fluorouracil, capecitabine, methotrexate sodium,ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine,5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine,pentostatin, fludarabine phosphate, and cladribine, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese.

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin. In someembodiments, the agent is a taxane. In certain embodiments, the agent ispaclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, orderivative of paclitaxel or docetaxel. In certain alternativeembodiments, the antimitotic agent comprises a vinca alkaloid, such asvincristine, binblastine, vinorelbine, or vindesine, or pharmaceuticallyacceptable salts, acids, or derivatives thereof.

As used herein, the term “immunotherapeutic agent” refers to a moleculethat can aid in the treatment of a disease by inducing, enhancing, orsuppressing an immune response in a cell, tissue, organ or subject.Examples of immunotherapeutic agents contemplated for use in combinationwith at least one metabolic reprogramming agent, at least two metabolicreprogramming agents, or at least three metabolic reprogramming agentsdescribed herein include, but are not limited to, immune checkpointmolecules (e.g., antibodies to immune checkpoint proteins), interleukins(e.g., IL-2, IL-7, IL-12, IL-15), cytokines (e.g., interferons, G-CSF,imiquimod), chemokines (e.g., CCL3, CCL26, CXCL7), vaccines (e.g.,peptide vaccines, dendritic cell (DC) vaccines, EGFRvIII vaccines,mesothilin vaccine, G-VAX, listeria vaccines), and adoptive T celltherapy including chimeric antigen receptor T cells (CAR T cells).

As used herein, “immunosuppressant agent” means an agent which may beused in immunotherapy to reduce or prevent an immune response in a cell,organ, tissue, or subject. Examples of immunosuppressant agentscontemplated for use in combination with at least one metabolicreprogramming agent, at least two metabolic reprogramming agents, or atleast three metabolic reprogramming agents include, without limitation,corticosteriods, calcineurin inhibitors, antiproliferative agents, SIPreceptor agonists, kinase inhibitors, monoclonal antilymphocyteantibodies and polyclonal antilymphocyte antibodies. Non-limitingexamples of corticosteroids include Prednisone (Deltasone® and Orasone®)and Methylprednisolone (SoluMedrol®). Non-limiting examples ofcalcineurin inhibitors include Cyclosporine (Cyclosporin A, SangCya,Sandimmune®, Neoral®, Gengraf®), ISA, Tx247, ABT-281, ASM 981 andTacrolimus (Prograf®, FK506). Non-limiting examples of antiproliferativeagents include Mycophenolate Mofetil (CellCept®), Azathioprene(Imuran®), and Sirolimus (Rapamune®). Non-limiting examples of SIPreceptor agonists include FTY 720 or analogues thereof. Non-limitingexamples of kinase inhibitors include mTOR kinase inhibitors, which arecompounds, proteins or antibodies that target, decrease or inhibit theactivity and/or function of members of the serine/threonine mTOR family.These include, without limitation, CCI-779, ABT578, SAR543, rapamycinand derivatives or analogs thereof, including40-O-(2-hydroxyethyl)-rapamycin, rapalogs, including AP23573, AP23464,AP23675 and AP23841 from Ariad, Everolimus (CERTICAN, RAD001), biolimus7, biolimus 9 and sirolimus (RAPAMUNE). Kinase inhibitors also includeprotein kinase C inhibitors, which include the compounds described thePCT publications WO 2005/097108 and WO 2005/068455, which are hereinincorporated by reference in their entireties. Non-limiting examples ofmonoclonal antilymphocyte antibodies include Muromonab-CD3 (OrthocloneOKT3®), Interleukin-2 Receptor Antagonist (Basiliximab, Simulect®), andDaclizumab (Zenapax®). Non-limiting examples of polyclonalantilymphocyte antibodies include Antithymocyte globulin-equine (Atgam®)and Antithymocyte globulin-rabbit (RATG, Thymoglobulin®). Otherimmunosuppressants include, without limitation, SERP-1, a serineprotease inhibitor produced by malignant rabbit fibroma virus (MRV) andmyxoma virus (MYX), described in US Patent Publication No. 2004/0029801,which is incorporated herein by reference.

Immunosuppressant agents can be classified according to their specificmolecular mode of action. The four main categories of immunosuppressantdrugs currently used in treating patients with transplanted organs arethe following. Cal cineurin inhibitors inhibit T-cell activation, thuspreventing T-cells from attacking the transplanted organ. Azathioprinesdisrupt the synthesis of DNA and RNA as well as the process of celldivision. Monoclonal antibodies inhibit the binding of interleukin-2,which in turn slows down the production of I-cells in the patient'simmune system. Corticosteroids suppress inflammation associated withtransplant rejection.

Immunosuppressants can also be classified according to the specificorgan that is transplanted. Basiliximab (Simulect) is also used incombination with such other drugs as cyclosporine and corticosteroids inkidney transplants. IL-2 blockers, including Simulect from Novartis,FK506 or CyA, MMF, prednisone or Rapamycin are also used in kidneytransplants. Daclizumab (Zenapax) is also used in combination with suchother drugs as cyclosporin and corticosteroids in kidney transplants.Similar drugs are used in heart transplants, but anti-lymphocyteglobulin (ALG) is often used instead of Simulect. Muromonab CD3(Orthoclone OKT3) is used along with cyclosporine in kidney, liver andheart transplants. Tacrolimus (Prograf) is used in liver and kidneytransplants. It is under study for bone marrow, heart, pancreas,pancreatic island cell and small bowel transplantation.

As used herein, “photodynamic therapy”, also known as photoradiationtherapy, phototherapy, and photochemotherapy, refers to a treatment thatuses photosensitizing agents in combination with light to kill cancercells. The photosensitizing agents kill cancer cells upon lightactivation.

As used herein, “proton therapy”, also known as proton beam therapy,refers to a treatment that uses a beam of protons to irradiate and killcancer cells.

As used herein, “anti-inflammatory agent” refers to an agent that may beused to prevent or reduce an inflammatory response or inflammation in acell, tissue, organ, or subject. Exemplary anti-inflammatory agentscontemplated for use in combination with at least one metabolicreprogramming agent, at least two metabolic reprogramming agents, or atleast three metabolic reprogramming agents include, without limitation,steroidal anti-inflammatory agents, a nonsteroidal anti-inflammatoryagent, or a combination thereof. In some embodiments, anti-inflammatoryagents include clobetasol, alclofenac, alclometasone dipropionate,algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenacsodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen,apazone, balsalazide disodium, bendazac, benoxaprofen, benzydaminehydrochloride, bromelains, broperamole, budesonide, carprofen,cicloprofen, cintazone, cliprofen, clobetasol propionate, clobetas onebutyrate, clopirac, cloticasone propionate, cormethasone acetate,cortodoxone, deflazacort, desonide, desoximetasone, dexamethasone,dexamethasone acetate, dexamethasone dipropionate, diclofenac potassium,diclofenac sodium, diflorasone diacetate, diflumidone sodium,diflunisal, difluprednate, diftalone, dimethyl sulfoxide, drocinonide,endrysone, enlimomab, enolicam sodium, epirizole, etodolac, etofenamate,felbinac, fenamole, fenbufen, fenclofenac, fenclorac, fendosal,fenpipalone, fentiazac, flazalone, fluazacort, flufenamic acid,flumizole, flunisolide acetate, flunixin, flunixin meglumine, fluocortinbutyl, fluorometholone acetate, fluquazone, flurbiprofen, fluretofen,fluticasone propionate, furaprofen, furobufen, halcinonide, halobetasolpropionate, halopredone acetate, ibufenac, ibuprofen, ibuprofenaluminum, ibuprofen piconol, ilonidap, indomethacin, indomethacinsodium, indoprofen, indoxole, intrazole, isoflupredone acetate,isoxepac, isoxicam, ketoprofen, lofemizole hydrochloride, lomoxicam,loteprednol etabonate, meclofenamate sodium, meclofenamic acid,meclorisone dibutyrate, mefenamic acid, mesalamine, meseclazone,methylprednisolone suleptanate, momiflumate, nabumetone, naproxen,naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein,orpanoxin, oxaprozin, oxyphenbutazone, paranyline hydrochloride,pentosan polysulfate sodium, phenbutazone sodium glycerate, pirfenidone,piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen,prednazate, prifelone, prodolic acid, proquazone, proxazole, proxazolecitrate, rimexolone, romazarit, salcolex, salnacedin, salsalate,sanguinarium chloride, seclazone, sermetacin, sudoxicam, sulindac,suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap,tenidap sodium, tenoxicam, tesicam, tesimide, tetrydamine, tiopinac,tixocortol pivalate, tolmetin, tolmetin sodium, triclonide,triflumidate, zidometacin, zomepirac sodium, aspirin (acetylsalicylicacid), salicylic acid, corticosteroids, glucocorticoids, tacrolimus,pimecorlimus, prodrugs thereof, co-drugs thereof, and combinationsthereof. The anti-inflammatory agent may also be a biological inhibitorof proinflammatory signaling molecules including antibodies to suchbiological inflammatory signaling molecules.

Exemplary neuroprotective agents include, without limitation, L-dopa,dopamine agonists (e.g., apomorphine, bromocriptine, pergolide,ropinirole, pramipexole, or cabergoline), adenosine A2a antagonists(Shah et al., Curr. Opin. Drug Discov. Devel. 13:466-80 (2010));serotonin receptor agonists; continuous-release levodopa (Sinemet CR®,MSD, Israel); continuous duodenal levodopa administration (Duodopa®,Abbott, UK); catechol-O-methyltransferase (COMT) inhibitors (e.g.,Stalevo®, Novartis Pharma, USA; entacapone (Comtan®, Novartis Pharma,USA)); tolcapone; coenzyme Q10, and/or MAO-B inhibitors (e.g.,Selegiline or Rasagiline). Additional neuroprotective agents aredescribed in, e.g., Hart et al., Mov. Disord. 24: 647-54 (2009).

As used herein, a “radiotherapeutic agent” refers to those agentsconventionally adopted in the therapeutic field of cancer treatment andincludes photons having enough energy for chemical bond ionization, suchas, for instance, alpha (a), beta ((3), and gamma (γ) rays fromradioactive nuclei as well as x-rays. The radiation may be high-LET(linear energy transfer) or low-LET. LET is the energy transferred perunit length of the distance. High LET is said to be densely ionizingradiation and Low LET is said to be sparsely ionizing radiation.Representative examples of high-LET are neutrons and alpha particles.Representative examples of low-LET are x-ray and gamma rays. Low LETradiation including both x-rays and grays is most commonly used forradiotherapy of cancer patients. The radiation may be used for externalradiation therapy that is usually given on an outpatient basis or forinternal radiation therapy that uses radiation that is placed very closeto or inside the tumor. In case of internal radiation therapy, theradiation source is usually sealed in a small holder called an implant.Implants may be in the form of thin wires, plastic tubes calledcatheters, ribbons, capsules, or seeds. The implant is put directly intothe body. Internal radiation therapy may require a hospital stay. Theionizing radiation source is provided as a unit dose of radiation and ispreferably an x-ray tube since it provides many advantages, such asconvenient adjustable dosing where the source may be easily turned onand off, minimal disposal problems, and the like. A unit dose ofradiation is generally measured in gray (Gy). The ionizing radiationsource may also comprise a radioisotope, such as a solid radioisotopicsource (e.g., wire, strip, pellet, seed, bead, or the like), or a liquidradioisotopic filled balloon. In the latter case, the balloon has beenspecially configured to prevent leakage of the radioisotopic materialfrom the balloon into the body lumen or blood stream. Still further, theionizing radiation source may comprise a receptacle in the catheter bodyfor receiving radioisotopic materials like pellets or liquids. Theradioisotopic material may be selected to emit α, β and γ. Usually, aand β radiations are preferred since they may be quickly absorbed by thesurrounding tissue and will not penetrate substantially beyond the wallof the body lumen being treated. Accordingly, incidental irradiation ofthe heart and other organs adjacent to the treatment region can besubstantially eliminated. The total number of units provided will be anamount determined to be therapeutically effective by one skilled intreatment using ionizing radiation. This amount will vary with thesubject and the type of malignancy or neoplasm being treated. The amountmay vary but a patient may receive a dosage of about 30-75 Gy overseveral weeks.

Exemplary radiotherapeutic agents contemplated for use in combinationwith at least one metabolic reprogramming agent, at least two metabolicreprogramming agents, or at least three metabolic reprogramming agentsinclude, factors that cause DNA damage, such as γ-rays, X-rays, and/orthe directed delivery of radioisotopes to tumor cells. Other forms ofDNA damaging factors are also contemplated, such as microwaves andUV-irradiation. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the target cell. In someembodiments, the radiotherapeutic agent is selected from the groupconsisting of ⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re,¹⁹⁹Au, ²¹¹At, ²¹²Pb, ²¹²B, ³²P and ³³P, ⁷¹Ge, ⁷⁷As, ¹⁰³Pb, ¹⁰⁵Rh, ¹¹¹Ag,¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Ln, ¹⁹¹Os, ¹⁹³MPt, ¹⁹⁷H, ⁴³K, ⁴³K,⁵²Fe, ⁷⁵Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁸¹Rb/⁸¹MKr, ⁸⁷MSr, ⁹⁹MTc, ¹¹¹In,¹¹³MIn, ¹²⁷Cs, ¹²⁹Cs, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶Bi, as described in U.S.Pat. No. 8,946,168, the entirety of which is incorporated herein byreference.

In some contexts, an agent described herein can be administered with anantigen (e.g., to induce an immune response). In some embodiments, anadjuvant can be used in combination with the antigen.

An agent described herein can also be used in combination with animaging agent. An agent (e.g., a metabolic reprogramming agent) can beattached to imaging agents for imaging and diagnosis of various diseasedorgans, tissues or cell types. The agent can be labeled or conjugated afluorophore or radiotracer for use as an imaging agent. Many appropriateimaging agents are known in the art, as are methods for their attachmentto agents (e.g., attaching an imaging agent to a proteins or peptidesusing metal chelate complexes, radioisotopes, fluorescent markers, orenzymes whose presence can be detected using a colorimetric markers(such as, but not limited to, urease, alkaline phosphatase,(horseradish) hydrogen peroxidase and glucose oxidase)). An agent mayalso be dual labeled with a radioisotope in order to combine imagingthrough nuclear approaches and be made into a unique cyclic structureand optimized for binding affinity and pharmacokinetics. Such agents canbe administered by any number of methods known to those of ordinaryskill in the art including, but not limited to, oral administration,inhalation, subcutaneous (sub-q), intravenous (I.V.), intraperitoneal(LP.), intramuscular (I.M.), intrathecal injection, or intratumoralinjection. The methods, compositions, and uses described herein can beused alone or in combination with other techniques, to diagnose accessand monitor and direct therapy of metabolic reprogramming disorders. Insome contexts, the imaging agent can be used for detecting and/ormonitoring tumors or sites of metastasis in a subject. For example, anagent (e.g., a metabolic reprogramming agent) can be administered invivo and monitored using an appropriate label. Exemplary methods fordetecting and/or monitoring an agent labeled with an imaging agent invivo include Gamma Scintigraphy, Positron Emission Tomography (PET),Single Photon Emission Computer Tomography (SPECT), Magnetic ResonanceImaging (MRI), X-ray, Computer Assisted X-ray Tomography (CT), NearInfrared Spectroscopy, and Ultrasound. These techniques provideinformation regarding detection of neoplastic involvement, particularlyof inaccessible nodes in subjects with malignant diseases. Knowledge onthe size of the node and the filling of nodes can also be instructive.For example, agents or compositions targeted to the lymph nodes indetection applications will contain suitable contrast or imaging agents,such as ferromagnetic materials like iron oxide, perfluorochemicals suchas perfluorooctylbromide, or gamma emitting radiolabels such asTechnetium-99m, Indium-111, Gallium-67, Thallium-201, Iodine-131, 125,or 123, positron emitting radiolabels, such as Fluorine-18, or thoseproduced by neutron activation, such as Samarium-153.

Imaging agents of use in the present disclosure include radioisotopesand dyes. Any conventional method according to radiolabeling which issuitable for labeling isotopes for in vivo use will be generallysuitable for labeling detection agents according to the disclosure.Internal detection procedures include intraoperative, intravascular orendoscopic, including laparoscopic, techniques, both surgically invasiveand noninvasive. For example, when detecting a lymph node, a highsignal-to-background ratio should to be achieved. Therapy also requiresa high absolute accretion of the therapeutic agent in the lymph node, aswell as a reasonably long duration of uptake and binding.

Suitable radioisotopes for the methods of the disclosure include:Actinium-225, Astatine-211, Iodine-123, Iodine-125, Iodine-126,Iodine-131, Iodine-133, Bismuth-212, Bromine-77, Indium-111,Indium-113m, Gallium-67, Gallium-68, Ruthenium-95, Ruthenium-97,Ruthenium-103, Ruthenium-105, Mercury-107, Mercury-203, Rhenium-186,Rhenium-188, Tellurium-121m, Tellurium-122m, Tellurium-125m,Thulium-165, Thulium-167, Thulium-168, Technetium-99m, Fluorine-18,Silver-111, Platinum-197, Palladium-109, Copper-67, Phosphorus-32,Phosphorus-33, Yttrium-90, Scandium-47, Samarium-153, Lutetium-177,Rhodium-105, Praseodymium-142, Praseodymium-143, Terbium-161,Holmium-166, Gold-199, Cobalt-57, Cobalt-58, Chromium-51, Iron-59,Selenium-75, Thallium-201, and Ytterbium-169. The most preferredradioisotope for use in the presently disclosed subject matter isTechnetium-99m. Preferably the radioisotope will emit a particle or rayin the 10-7,000 keV range, more preferably in the 50-1,500 keV range,and most preferably in the 80-250 keV range.

Isotopes preferred for external imaging include: Iodine-123, Iodine-131,Indium-111, Gallium-67, Ruthenium-97, Technetium-99m, Cobalt-57,Cobalt-58, Chromium-51, Iron-59, Selenium-75, Thallium-201, andYtterbium-169. Technetium-99m is the most preferred radioisotope forexternal imaging in the disclosure.

Isotopes most preferred for internal detection include: Iodine-125,Iodine-123, Iodine-131, Indium-111, Technetium-99m and Gallium-67.Technetium-99m is the most preferred isotope for internal detection.

III. Uses of Metabolic Reprogramming Agents

The presently disclosed subject matter contemplates the use of at leastone, at least two, or at least three metabolic reprogramming agents thatdecrease activity of at least one metabolic pathway selected from thegroup consisting of glutamine metabolism, glycolysis, and fatty acidsynthesis, alone, or optionally together with one or more additionaltherapeutic agents described herein. Accordingly, in an aspect thepresently disclosed subject matter involves the use of at least onemetabolic reprogramming agent that decreases activity of at least onemetabolic pathway selected from the group consisting of glutaminemetabolism, glycolysis, and fatty acid synthesis for treating acondition, disease, or disorder that involves (i) metabolicallyreprogrammed cells whose activation, function, growth, proliferation,and/or survival depends on increased activity of at least one metabolicpathway selected from the group consisting of glutamine metabolism,glycolysis, and fatty acid synthesis or (ii) at least one of aberrantand/or excessive glutamine metabolism, aberrant and/or excessiveglycolysis, or aberrant and/or excessive fatty acid synthesis.

In some embodiments, the presently disclosed subject matter involves theuse of at least two metabolic reprogramming agents. In some embodiments,the presently disclosed subject matter involves the use of at leastthree metabolic reprogramming agents.

In some aspects, the presently disclosed subject matter involves the useof at least one metabolic reprogramming agent that decreases glutaminemetabolism as an immunotherapy to treat a cancer. In other aspects, thepresently disclosed subject matter involves the use of at least onemetabolic reprogramming agent that decreases glutamine metabolism as animmunotherapy in combination with an additional immunotherapy to treat acancer. Examples of additional immunotherapy contemplated for use incombination with the at least one metabolic reprogramming agent include,without limitation, checkpoint blockade, adoptive cellular therapy,CAR-T cell therapy, marrow-infiltrating lymphocytes, A2aR blockade, KIRblockade, vaccines (e.g., tumor vaccines), passive immunotherapyantibodies, and combinations thereof.

In an aspect, the presently disclosed subject matter involves the use ofan effective amount of at least one metabolic reprogramming agent thatdecreases glutamine metabolism to treat lymphoma in a subject in needthereof.

In an aspect, the presently disclosed subject matter involves the use ofan effective amount of at least one metabolic reprogramming agent thatdecreases glutamine metabolism to treat melanoma in a subject in needthereof.

In some embodiments, a use described herein further comprises using aneffective amount of at least one metabolic reprogramming agent thatdecreases glycolysis. In some embodiments, a use described hereinfurther comprises uses an effective amount of at least one metabolicreprogramming agent that increases fatty acid oxidation.

IV. Pharmaceutical Compositions Comprising Metabolic ReprogrammingAgents

The presently disclosed subject matter also contemplates pharmaceuticalcompositions comprising one or more metabolic reprogramming agents forthe treatment of certain conditions, diseases, and/or disordersinvolving metabolically reprogrammed cells. In some embodiments, thepresently disclosed methods comprise the use of the presently disclosedmetabolic reprogramming agents for the manufacture of a medicament forthe treatment of certain conditions, diseases, and/or disorders involvemetabolically reprogrammed cells. The disclosure contemplates variouspharmaceutical compositions comprising at least one, at least two, andor at least three metabolic reprogramming agents.

Accordingly, in an aspect the presently disclosed subject matterprovides a pharmaceutical composition comprising an effective amount ofat least one, at least two, or at least three metabolic reprogrammingagents that decrease the activity of at least one metabolic pathwayselected from the group consisting of glutamine metabolism, glycolysis,and fatty acid synthesis, and a pharmaceutically acceptable carrier,diluent, or excipient.

In some aspects, the presently disclosed subject matter provides apharmaceutical composition comprising at least one metabolicreprogramming agent that decreases glutamine metabolism as animmunotherapy to treat a cancer, and a pharmaceutically acceptablecarrier, diluent, or excipient. It should be appreciated that additionalforms of immunotherapy are contemplated for use in combination with thepharmaceutical composition comprising at least one metabolicreprogramming agent, such as checkpoint blockade, adoptive cellulartherapy, CAR-T cell therapy, marrow-infiltrating lymphocytes, A2aRblockade, KIR blockade, vaccines (e.g., tumor vaccines), passiveimmunotherapy antibodies, and combinations thereof.

In some embodiments, the metabolic reprogramming composition comprisesone or more additional therapeutic agents described herein. Generally,the presently disclosed compositions (e.g., comprising at least onemetabolic reprogramming agent) can be administered to a subject fortherapy by any suitable route of administration, including orally,nasally, transmucosally, ocularly, rectally, intravaginally,parenterally, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intra-articular, intra-sternal, intra-synovial,intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal,intraocular injections, intratumoral injections, intracisternally,topically, as by powders, ointments or drops (including eyedrops),including buccally and sublingually, transdermally, through aninhalation spray, or other modes of delivery known in the art.

The phrases “systemic administration”, “administered systemically”,“peripheral administration” and “administered peripherally” as usedherein mean the administration of compositions comprising at least onemetabolic reprogramming agent, such that it enters the patient's systemand, thus, are subject to metabolism and other like processes, forexample, subcutaneous administration.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intarterial, intrathecal,intracapsular, intraorbital, intraocular, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The presently disclosed pharmaceutical compositions can be manufacturedin a manner known in the art, e.g. by means of conventional mixing,dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

In some embodiments, the presently disclosed pharmaceutical compositionscan be administered by rechargeable or biodegradable devices. Forexample, a variety of slow-release polymeric devices have been developedand tested in vivo for the controlled delivery of drugs, includingproteinacious biopharmaceuticals. Suitable examples of sustained releasepreparations include semipermeable polymer matrices in the form ofshaped articles, e.g., films or microcapsules. Sustained releasematrices include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919; EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers 22:547, 1983), poly(2-hydroxyethyl-methacrylate) (Langer et al. (1981) J. Biomed. Mater.Res. 15:167; Langer (1982), Chem. Tech. 12:98), ethylene vinyl acetate(Langer et al. (1981) J. Biomed. Mater. Res. 15:167), orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988A). Sustained releasecompositions also include liposomally entrapped compositions comprisingat least one metabolic reprogramming agent which can be prepared bymethods known in the art (Epstein et al. (1985) Proc. Natl. Acad. Sci.U.S.A. 82:3688; Hwang et al. (1980) Proc. Natl. Acad. Sci. U.S.A.77:4030; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324A).Ordinarily, the liposomes are of the small (about 200-800 angstroms)unilamelar type in which the lipid content is greater than about 30 mol% cholesterol, the selected proportion being adjusted for the optimaltherapy. Such materials can comprise an implant, for example, forsustained release of the presently disclosed compositions, which, insome embodiments, can be implanted at a particular, pre-determinedtarget site.

In another embodiment, the presently disclosed pharmaceuticalcompositions may comprise PEGylated therapeutics (e.g., PEGylatedantibodies). PEGylation is a well established and validated approach forthe modification of a range of antibodies, proteins, and peptides andinvolves the attachment of polyethylene glycol (PEG) at specific sitesof the antibodies, proteins, and peptides (Chapman (2002) Adv. DrugDeliv. Rev. 54:531-545). Some effects of PEGylation include: (a)markedly improved circulating half-lives in vivo due to either evasionof renal clearance as a result of the polymer increasing the apparentsize of the molecule to above the glomerular filtration limit, and/orthrough evasion of cellular clearance mechanisms; (b) improvedpharmacokinetics; (c) improved solubility—PEG has been found to besoluble in many different solvents, ranging from water to many organicsolvents, such as toluene, methylene chloride, ethanol and acetone; (d)PEGylated antibody fragments can be concentrated to 200 mg/ml, and theability to do so opens up formulation and dosing options, such assubcutaneous administration of a high protein dose; this is in contrastto many other therapeutic antibodies which are typically administeredintravenously; (e) enhanced proteolytic resistance of the conjugatedprotein (Cunningham-Rundles et. al. (1992) J. Immunol. Meth.152:177-190); (f) improved bioavailability via reduced losses atsubcutaneous injection sites; (g) reduced toxicity has been observed;for agents where toxicity is related to peak plasma level, a flatterpharmacokinetic profile achieved by sub-cutaneous administration ofPEGylated protein is advantageous; proteins that elicit an immuneresponse which has toxicity consequences may also benefit as a result ofPEGylation; and (h) improved thermal and mechanical stability of thePEGylated molecule.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of compositions comprising at least one metabolicreprogramming agent. For injection, the presently disclosedpharmaceutical compositions can be formulated in aqueous solutions, forexample, in some embodiments, in physiologically compatible buffers,such as Hank's solution, Ringer's solution, or physiologically bufferedsaline. Aqueous injection suspensions can contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions ofcompositions include fatty oils, such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension also can contain suitable stabilizers oragents that increase the solubility of the compositions comprising atleast one metabolic reprogramming agent to allow for the preparation ofhighly concentrated solutions.

For nasal or transmucosal administration generally, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

Additional ingredients can be added to compositions for topicaladministration, as long as such ingredients are pharmaceuticallyacceptable and not deleterious to the epithelial cells or theirfunction. Further, such additional ingredients should not adverselyaffect the epithelial penetration efficiency of the composition, andshould not cause deterioration in the stability of the composition. Forexample, fragrances, opacifiers, antioxidants, gelling agents,stabilizers, surfactants, emollients, coloring agents, preservatives,buffering agents, and the like can be present. The pH of the presentlydisclosed topical composition can be adjusted to a physiologicallyacceptable range of from about 6.0 to about 9.0 by adding bufferingagents thereto such that the composition is physiologically compatiblewith a subject's skin.

Regardless of the route of administration selected, the presentlydisclosed compositions are formulated into pharmaceutically acceptabledosage forms, such as described herein or by other conventional methodsknown to those of skill in the art.

In general, the “effective amount” or “therapeutically effective amount”of an active agent or drug delivery device refers to the amountnecessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof an agent or device may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the composition of theencapsulating matrix, the target tissue, and the like.

The term “combination” is used in its broadest sense and means that asubject is administered at least two agents. More particularly, the term“in combination” refers to the concomitant administration of two (ormore) active agents for the treatment of a, e.g., single disease state.As used herein, the active agents may be combined and administered in asingle dosage form, may be administered as separate dosage forms at thesame time, or may be administered as separate dosage forms that areadministered alternately or sequentially on the same or separate days.In one embodiment of the presently disclosed subject matter, the activeagents are combined and administered in a single dosage form. In anotherembodiment, the active agents are administered in separate dosage forms(e.g., wherein it is desirable to vary the amount of one but not theother). The single dosage form may include additional active agents forthe treatment of the disease state.

Further, the presently disclosed compositions can be administered aloneor in combination with adjuvants that enhance stability of the agents,facilitate administration of pharmaceutical compositions containing themin certain embodiments, provide increased dissolution or dispersion,increase activity, provide adjuvant therapy, and the like, includingother active ingredients. Advantageously, such combination therapiesutilize lower dosages of the conventional therapeutics, thus avoidingpossible toxicity and adverse side effects incurred when those agentsare used as monotherapies.

The timing of administration of at least one metabolic reprogrammingagent can be varied so long as the beneficial effects of the combinationof these agents are achieved. Accordingly, the phrase “in combinationwith” refers to the administration of at least one metabolicreprogramming agent, at least two metabolic reprogramming agents, or atleast three metabolic reprogramming agents, and optionally additionalagents either simultaneously, sequentially, or a combination thereof.Therefore, a subject administered a combination of at least one, atleast two, or at least three metabolic reprogramming agents, andoptionally additional agents can receive at least one metabolicreprogramming agent, at least two metabolic reprogramming agents, and atleast three metabolic reprogramming agents, and optionally additionalagents at the same time (i.e., simultaneously) or at different times(i.e., sequentially, in either order, on the same day or on differentdays), so long as the effect of the combination of all agents isachieved in the subject.

When administered sequentially, the agents can be administered within 1,5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In otherembodiments, agents administered sequentially, can be administeredwithin 1, 2, 3, 4, 5, 10, 15, 20 or more days of one another. Where theagents are administered simultaneously, they can be administered to thesubject as separate pharmaceutical compositions, each comprising eitherat least one metabolic reprogramming agent, at least two metabolicreprogramming agents, or at least three metabolic reprogramming agents,and optionally additional agents, or they can be administered to asubject as a single pharmaceutical composition comprising all agents.

When administered in combination, the effective concentration of each ofthe agents to elicit a particular biological response may be less thanthe effective concentration of each agent when administered alone,thereby allowing a reduction in the dose of one or more of the agentsrelative to the dose that would be needed if the agent was administeredas a single agent. The effects of multiple agents may, but need not be,additive or synergistic. The agents may be administered multiple times.

In some embodiments, when administered in combination, the two or moreagents can have a synergistic effect. As used herein, the terms“synergy,” “synergistic,” “synergistically” and derivations thereof,such as in a “synergistic effect” or a “synergistic combination” or a“synergistic composition” refer to circumstances under which thebiological activity of a combination of an agent and at least oneadditional therapeutic agent is greater than the sum of the biologicalactivities of the respective agents when administered individually.

Synergy can be expressed in terms of a “Synergy Index (SI),” whichgenerally can be determined by the method described by F. C. Kull et al.Applied Microbiology 9, 538 (1961), from the ratio determined by:

Q _(a) Q _(A) +Q _(b) Q _(B)=Synergy Index (SI)

wherein:

Q_(A) is the concentration of a component A, acting alone, whichproduced an end point in relation to component A;

Q_(a) is the concentration of component A, in a mixture, which producedan end point;

Q_(B) is the concentration of a component B, acting alone, whichproduced an end point in relation to component B; and

Q_(b) is the concentration of component B, in a mixture, which producedan end point.

Generally, when the sum of Q_(a)/Q_(A) and Q_(b)/Q_(B) is greater thanone, antagonism is indicated. When the sum is equal to one, additivityis indicated. When the sum is less than one, synergism is demonstrated.The lower the SI, the greater the synergy shown by that particularmixture. Thus, a “synergistic combination” has an activity higher thatwhat can be expected based on the observed activities of the individualcomponents when used alone. Further, a “synergistically effectiveamount” of a component refers to the amount of the component necessaryto elicit a synergistic effect in, for example, another therapeuticagent present in the composition.

In another aspect, the presently disclosed subject matter provides apharmaceutical composition including at least one metabolicreprogramming agent, at least two metabolic reprogramming agents, atleast three metabolic reprogramming agents, and optionally additionalagents, alone or in combination with one or more additional therapeuticagents in admixture with a pharmaceutically acceptable excipient.

More particularly, the presently disclosed subject matter provides apharmaceutical composition comprising at least one metabolicreprogramming agent, at least two metabolic reprogramming agents, atleast three metabolic reprogramming agents, and optionally additionalagents, and a pharmaceutically acceptable carrier.

In therapeutic and/or diagnostic applications, the compounds of thedisclosure can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20^(th) ed.) Lippincott, Williams and Wilkins(2000).

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe disclosure to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya subject (e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure also maybe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances, such as saline; preservatives, suchas benzyl alcohol; absorption promoters; and fluorocarbons.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.Generally, the compounds according to the disclosure are effective overa wide dosage range. For example, in the treatment of adult humans,dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg perday, and from 5 to 40 mg per day are examples of dosages that may beused. A non-limiting dosage is 10 to 30 mg per day. The exact dosagewill depend upon the route of administration, the form in which thecompound is administered, the subject to be treated, the body weight ofthe subject to be treated, and the preference and experience of theattending physician.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The term “instructing” a patient as used herein means providingdirections for applicable therapy, medication, treatment, treatmentregimens, and the like, by any means, but preferably in writing.Instructing can be in the form of prescribing a course of treatment, orcan be in the form of package inserts or other written promotionalmaterial. Accordingly, aspects of the presently disclosed subject matterinclude instructing a patient to receive a method of treatment or use anagent to treat a metabolic reprogramming disorder described herein.

The term “promoting” as used herein means offering, advertising,selling, or describing a particular drug, combination of drugs, ortreatment modality, by any means, including writing, such as in the formof package inserts. Promoting herein refers to promotion of a metabolicreprogramming agent for an indication, where such promoting isauthorized by the Food and Drug Administration (FDA) as having beendemonstrated to be associated with statistically significant therapeuticefficacy and acceptable safety in a population of subjects. In someembodiments, promoting is not authorized by the Food and DrugAdministration (FDA) (or other health regulatory agency, such as theEuropean Medicines Agency (EMA), and promoting is for an off-label use.Accordingly, aspects of the presently disclosed subject matter includepromoting a method of treatment or use described herein.

V. General Definitions

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

While the following terms in relation to compounds of Formula (I) arebelieved to be well understood by one of ordinary skill in the art, thefollowing definitions are set forth to facilitate explanation of thepresently disclosed subject matter. These definitions are intended tosupplement and illustrate, not preclude, the definitions that would beapparent to one of ordinary skill in the art upon review of the presentdisclosure.

The terms substituted, whether preceded by the term “optionally” or not,and substituent, as used herein, refer to the ability, as appreciated byone skilled in this art, to change one functional group for anotherfunctional group on a molecule, provided that the valency of all atomsis maintained. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The substituents also may be further substituted (e.g., anaryl group substituent may have another substituent off it, such asanother aryl group, which is further substituted at one or morepositions).

Where substituent groups or linking groups are specified by theirconventional chemical formulae, written from left to right, they equallyencompass the chemically identical substituents that would result fromwriting the structure from right to left, e.g., —CH₂O— is equivalent to—OCH₂—; —C(═O)O— is equivalent to —OC(═O)—; —OC(═O)NR— is equivalent to—NRC(═O)O—, and the like.

When the term “independently selected” is used, the substituents beingreferred to (e.g., R groups, such as groups R₁, R₂, and the like, orvariables, such as “m” and “n”), can be identical or different. Forexample, both R₁ and R₂ can be substituted alkyls, or R₁ can be hydrogenand R2 can be a substituted alkyl, and the like.

The terms “a,” “an,” or “a(n),” when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

A named “R” or group will generally have the structure that isrecognized in the art as corresponding to a group having that name,unless specified otherwise herein. For the purposes of illustration,certain representative “R” groups as set forth above are defined below.

Descriptions of compounds of the present disclosure are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

Unless otherwise explicitly defined, a “substituent group,” as usedherein, includes a functional group selected from one or more of thefollowing moieties, which are defined herein:

The term hydrocarbon, as used herein, refers to any chemical groupcomprising hydrogen and carbon. The hydrocarbon may be substituted orunsubstituted. As would be known to one skilled in this art, allvalencies must be satisfied in making any substitutions. The hydrocarbonmay be unsaturated, saturated, branched, unbranched, cyclic, polycyclic,or heterocyclic. Illustrative hydrocarbons are further defined hereinbelow and include, for example, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, andthe like.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedchain, acyclic or cyclic hydrocarbon group, or combination thereof,which may be fully saturated, mono- or polyunsaturated and can includedi- and multivalent groups, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons, including 1, 2, 3, 4, 5, 6, 7,8, 9, and 10 carbons). In particular embodiments, the term “alkyl”refers to C₁₋₂₀ inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partiallyand in some cases fully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon radicals derived from a hydrocarbon moiety containingbetween one and twenty carbon atoms by removal of a single hydrogenatom.

Representative saturated hydrocarbon groups include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n-hexyl,sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers thereof.

“Branched” refers to an alkyl group in which a lower alkyl group, suchas methyl, ethyl or propyl, is attached to a linear alkyl chain. “Loweralkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e.,a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higheralkyl” refers to an alkyl group having about 10 to about 20 carbonatoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.In certain embodiments, “alkyl” refers, in particular, to C₁₋₈straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C₁₋₈ branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon group, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen,phosphorus, and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂₅—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N-OCH₃, —CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to twoor three heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

As described above, heteroalkyl groups, as used herein, include thosegroups that are attached to the remainder of the molecule through aheteroatom, such as —C(O)NR′, —NR′R″, —OR′, —SR, —S(O)R, and/or—S(O₂)R′. Where “heteroalkyl” is recited, followed by recitations ofspecific heteroalkyl groups, such as —NR′R or the like, it will beunderstood that the terms heteroalkyl and —NR′R″ are not redundant ormutually exclusive. Rather, the specific heteroalkyl groups are recitedto add clarity. Thus, the term “heteroalkyl” should not be interpretedherein as excluding specific heteroalkyl groups, such as —NR′R″ or thelike.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, unsubstituted alkyl,substituted alkyl, aryl, or substituted aryl, thus providing aheterocyclic group. Representative monocyclic cycloalkyl rings includecyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl ringsinclude adamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl, and fused ring systems, such as dihydro- andtetrahydronaphthalene, and the like.

The term “cycloalkylalkyl,” as used herein, refers to a cycloalkyl groupas defined hereinabove, which is attached to the parent molecular moietythrough an alkyl group, also as defined above. Examples ofcycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

The terms “cycloheteroalkyl” or “heterocycloalkyl” refer to anon-aromatic ring system, unsaturated or partially unsaturated ringsystem, such as a 3- to 10-member substituted or unsubstitutedcycloalkyl ring system, including one or more heteroatoms, which can bethe same or different, and are selected from the group consisting ofnitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si),and optionally can include one or more double bonds.

The cycloheteroalkyl ring can be optionally fused to or otherwiseattached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbonrings. Heterocyclic rings include those having from one to threeheteroatoms independently selected from oxygen, sulfur, and nitrogen, inwhich the nitrogen and sulfur heteroatoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. In certainembodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or7-membered ring or a polycyclic group wherein at least one ring atom isa heteroatom selected from 0, S, and N (wherein the nitrogen and sulfurheteroatoms may be optionally oxidized), including, but not limited to,a bi- or tri-cyclic group, comprising fused six-membered rings havingbetween one and three heteroatoms independently selected from theoxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfurheteroatoms may be optionally oxidized, (iii) the nitrogen heteroatommay optionally be quaternized, and (iv) any of the above heterocyclicrings may be fused to an aryl or heteroaryl ring. Representativecycloheteroalkyl ring systems include, but are not limited topyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl,morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and thelike.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

An unsaturated alkyl group is one having one or more double bonds ortriple bonds. Examples of unsaturated alkyl groups include, but are notlimited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl.”

More particularly, the term “alkenyl” as used herein refers to amonovalent group derived from a C₁₋₂₀ inclusive straight or branchedhydrocarbon moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen molecule. Alkenyl groups include, forexample, ethenyl (i.e., vinyl), propenyl, butenyl,1-methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl, allenyl, andbutadienyl.

The term “cycloalkenyl” as used herein refers to a cyclic hydrocarboncontaining at least one carbon-carbon double bond. Examples ofcycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

The term “alkynyl” as used herein refers to a monovalent group derivedfrom a straight or branched C1-20 hydrocarbon of a designed number ofcarbon atoms containing at least one carbon-carbon triple bond. Examplesof “alkynyl” include ethynyl, 2-propynyl (propargyl), 1-propynyl,pentynyl, hexynyl, and heptynyl groups, and the like.

The term “alkylene” by itself or a part of another substituent refers toa straight or branched bivalent aliphatic hydrocarbon group derived froman alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. The alkylene group can be straight, branched or cyclic. Thealkylene group also can be optionally unsaturated and/or substitutedwith one or more “alkyl group substituents.” There can be optionallyinserted along the alkylene group one or more oxygen, sulfur orsubstituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl aspreviously described. Exemplary alkylene groups include methylene(—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH₂)₃-); cyclohexylene(—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; —CH₂CH₂CH₂CH₂—, —CH₂CH═CHCH₂—,—CH₂C_(S)CCH₂—, —CH₂CH₂CH(CH₂CH₂CH₃)CH₂—, —(CH₂)_(q)—N(R)—(CH₂)_(r)—,wherein each of q and r is independently an integer from 0 to about 20,e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl(—O—CH₂—O—); and ethylenedioxyl (—O— (CH₂)₂—O—). An alkylene group canhave about 2 to about 3 carbon atoms and can further have 6-20 carbons.Typically, an alkyl (or alkylene) group will have from 1 to 24 carbonatoms, with those groups having 10 or fewer carbon atoms being someembodiments of the present disclosure. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The term “heteroalkylene” by itself or as part of another substituentmeans a divalent group derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms also can occupy either or both of thechain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)OR′— represents both —C(O)OR′—and —R′OC(O)—.

The term “aryl” means, unless otherwise stated, an aromatic hydrocarbonsub stituent that can be a single ring or multiple rings (such as from 1to 3 rings), which are fused together or linked covalently. The term“heteroaryl” refers to aryl groups (or rings) that contain from one tofour heteroatoms (in each separate ring in the case of multiple rings)selected from N, O, and S, wherein the nitrogen and sulfur atoms areoptionally oxidized, and the nitrogen atom(s) are optionallyquaternized. A heteroaryl group can be attached to the remainder of themolecule through a carbon or heteroatom. Non-limiting examples of aryland heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryland heteroaryl ring systems are selected from the group of acceptablesubstituents described below. The terms “arylene” and “heteroarylene”refer to the divalent forms of aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the terms “arylalkyl” and“heteroarylalkyl” are meant to include those groups in which an aryl orheteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl,pyridylmethyl, furylmethyl, and the like) including those alkyl groupsin which a carbon atom (e.g., a methylene group) has been replaced by,for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,3-(1-naphthyloxy)propyl, and the like). However, the term “haloaryl,” asused herein is meant to cover only aryls substituted with one or morehalogens.

Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specificnumber of members (e.g. “3 to 7 membered”), the term “member” refers toa carbon or heteroatom.

Further, a structure represented generally by the formula:

as used herein refers to a ring structure, for example, but not limitedto a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and thelike, aliphatic and/or aromatic cyclic compound, including a saturatedring structure, a partially saturated ring structure, and an unsaturatedring structure, comprising a substituent R group, wherein the R groupcan be present or absent, and when present, one or more R groups caneach be substituted on one or more available carbon atoms of the ringstructure. The presence or absence of the R group and number of R groupsis determined by the value of the variable “n,” which is an integergenerally having a value ranging from 0 to the number of carbon atoms onthe ring available for substitution. Each R group, if more than one, issubstituted on an available carbon of the ring structure rather than onanother R group. For example, the structure above where n is 0 to 2would comprise compound groups including, but not limited to:

and the like.

A dashed line representing a bond in a cyclic ring structure indicatesthat the bond can be either present or absent in the ring. That is, adashed line representing a bond in a cyclic ring structure indicatesthat the ring structure is selected from the group consisting of asaturated ring structure, a partially saturated ring structure, and anunsaturated ring structure.

The symbol (

) denotes the point of attachment of a moiety to the remainder of themolecule.

When a named atom of an aromatic ring or a heterocyclic aromatic ring isdefined as being “absent,” the named atom is replaced by a direct bond.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl”, “aryl,” “heteroaryl,” “phosphonate,” and “sulfonate”as well as their divalent derivatives) are meant to include bothsubstituted and unsubstituted forms of the indicated group. Optionalsubstituents for each type of group are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative groups (including those groups oftenreferred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, 502 NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″, —OC(O)NR′R″,—NR″ C(O)R′, —NR′—C(O)NR″R′″, —NR” C(O)OR′, —NR—C(NR′R″)═NR′″, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂ in a number ranging fromzero to (2m′+1), where m′ is the total number of carbon atoms in suchgroups. R′, R″, R′″ and R″″ each may independently refer to hydrogen,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, orarylalkyl groups. As used herein, an “alkoxy” group is an alkyl attachedto the remainder of the molecule through a divalent oxygen. When acompound of the disclosure includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. When R′and R″ are attached to the same nitrogen atom, they can be combined withthe nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for alkyl groups above, exemplarysubstituents for aryl and heteroaryl groups (as well as their divalentderivatives) are varied and are selected from, for example: halogen,—OR′, —NR′R″, —SR′, —SiR′R″R″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″-S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxo, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on aromatic ring system; and where R′, R″, R′″ and R″″ maybe independently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. When a compound of the disclosure includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CRR′— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4.

One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CRR′)_(s)—X′— (C″R′″)_(d)—, where sand d are independently integers of from 0 to 3, and X′ is —O—, —NR′—,—S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—. The substituents R, R′, R″ and R′″may be independently selected from hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

As used herein, the term “acyl” refers to an organic acid group whereinthe —OH of the carboxyl group has been replaced with another substituentand has the general formula RC(═O)—, wherein R is an alkyl, alkenyl,alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic groupas defined herein). As such, the term “acyl” specifically includesarylacyl groups, such as a 2-(furan-2-yl)acetyl)- and a 2-phenylacetylgroup. Specific examples of acyl groups include acetyl and benzoyl. Acylgroups also are intended to include amides, —RC(═O)NR′, esters,—RC(═O)OR′, ketones, —RC(═O)R′, and aldehydes, —RC(═O)H.

The terms “alkoxyl” or “alkoxy” are used interchangeably herein andrefer to a saturated (i.e., alkyl-O—) or unsaturated (i.e., alkenyl-O—and alkynyl-O—) group attached to the parent molecular moiety through anoxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are aspreviously described and can include C₁₋₂₀ inclusive, linear, branched,or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including,for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl,sec-butoxyl, tert-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, andthe like.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether,for example, a methoxyethyl or an ethoxymethyl group.

“Aryloxyl” refers to an aryl-O— group wherein the aryl group is aspreviously described, including a substituted aryl. The term “aryloxyl”as used herein can refer to phenyloxyl or hexyloxyl, and alkyl,substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are aspreviously described, and included substituted aryl and substitutedalkyl. Exemplary aralkyl groups include benzyl, phenylethyl, andnaphthylmethyl.

“Aralkyloxyl” refers to an aralkyl-O— group wherein the aralkyl group isas previously described. An exemplary aralkyloxyl group is benzyloxyl,i.e., C₆H₅—CH₂—O—. An aralkyloxyl group can optionally be substituted.

“Alkoxycarbonyl” refers to an alkyl-O—C(═O)— group. Exemplaryalkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,butyloxycarbonyl, and tert-butyloxycarbonyl.

“Aryloxycarbonyl” refers to an aryl-O—C(═O)— group. Exemplaryaryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—C(═O)— group. An exemplaryaralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an amide group of the formula —C(═O)NH₂.

“Alkylcarbamoyl” refers to a R′RN—C(═O)— group wherein one of R and R′is hydrogen and the other of R and R′ is alkyl and/or substituted alkylas previously described. “Dialkylcarbamoyl” refers to a R′RN—C(═O)—group wherein each of R and R′ is independently alkyl and/or substitutedalkyl as previously described.

The term carbonyldioxyl, as used herein, refers to a carbonate group ofthe formula —O—C(═O)—OR.

“Acyloxyl” refers to an acyl-O— group wherein acyl is as previouslydescribed.

The term “amino” refers to the —NH₂ group and also refers to a nitrogencontaining group as is known in the art derived from ammonia by thereplacement of one or more hydrogen radicals by organic radicals. Forexample, the terms “acylamino” and “alkylamino” refer to specificN-substituted organic radicals with acyl and alkyl substituent groupsrespectively.

An “aminoalkyl” as used herein refers to an amino group covalently boundto an alkylene linker. More particularly, the terms alkylamino,dialkylamino, and trialkylamino as used herein refer to one, two, orthree, respectively, alkyl groups, as previously defined, attached tothe parent molecular moiety through a nitrogen atom. The term alkylaminorefers to a group having the structure —NHR′ wherein R′ is an alkylgroup, as previously defined; whereas the term dialkylamino refers to agroup having the structure —NR′R″, wherein R′ and R″ are eachindependently selected from the group consisting of alkyl groups. Theterm trialkylamino refers to a group having the structure —NR′R″R′″,wherein R′, R″, and R″ are each independently selected from the groupconsisting of alkyl groups. Additionally, R′, R″, and/or R′″ takentogether may optionally be —(CH₂)_(k)— where k is an integer from 2 to6. Examples include, but are not limited to, methylamino, dimethylamino,ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino,isopropylamino, piperidino, trimethylamino, and propylamino.

The amino group is —NR′R″, wherein R′ and R″ are typically selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

The terms alkylthioether and thioalkoxyl refer to a saturated (i.e.,alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) groupattached to the parent molecular moiety through a sulfur atom. Examplesof thioalkoxyl moieties include, but are not limited to, methylthio,ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

“Acylamino” refers to an acyl-NH— group wherein acyl is as previouslydescribed. “Aroylamino” refers to an aroyl-NH— group wherein aroyl is aspreviously described.

The term “carbonyl” refers to the —C(═O)— group, and can include analdehyde group represented by the general formula R—C(═O)H.

The term “carboxyl” refers to the —COOH group. Such groups also arereferred to herein as a “carboxylic acid” moiety.

The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups. Additionally, terms, such as“haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. Forexample, the term “halo(C₁-C₄)alkyl” is mean to include, but not belimited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “hydroxyl” refers to the —OH group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OHgroup.

The term “mercapto” refers to the —SH group.

The term “oxo” as used herein means an oxygen atom that is double bondedto a carbon atom or to another element.

The term “nitro” refers to the —NO₂ group.

The term “thio” refers to a compound described previously herein whereina carbon or oxygen atom is replaced by a sulfur atom.

The term “sulfate” refers to the —SO₄ group.

The term thiohydroxyl or thiol, as used herein, refers to a group of theformula —SH.

More particularly, the term “sulfide” refers to compound having a groupof the formula —SR.

The term “sulfone” refers to compound having a sulfonyl group —S(O₂)R.

The term “sulfoxide” refers to a compound having a sulfinyl group —S(O)R

The term ureido refers to a urea group of the formula —NH—CO—NH₂.

Throughout the specification and claims, a given chemical formula orname shall encompass all tautomers, congeners, and optical- andstereoisomers, as well as racemic mixtures where such isomers andmixtures exist.

Certain compounds of the present disclosure may possess asymmetriccarbon atoms (optical or chiral centers) or double bonds; theenantiomers, racemates, diastereomers, tautomers, geometric isomers,stereoisometric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)- or, as D- or L- for amino acids, andindividual isomers are encompassed within the scope of the presentdisclosure. The compounds of the present disclosure do not include thosewhich are known in art to be too unstable to synthesize and/or isolate.The present disclosure is meant to include compounds in racemic,scalemic, and optically pure forms. Optically active (R)- and (S)-, orD- and L-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques. When the compoundsdescribed herein contain olefenic bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of thedisclosure.

It will be apparent to one skilled in the art that certain compounds ofthis disclosure may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the disclosure. The term“tautomer,” as used herein, refers to one of two or more structuralisomers which exist in equilibrium and which are readily converted fromone isomeric form to another.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures with the replacement of a hydrogen by a deuterium or tritium,or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbon are withinthe scope of this disclosure.

The compounds of the present disclosure may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present disclosure, whether radioactive or not, are encompassedwithin the scope of the present disclosure.

The compounds of the present disclosure may exist as salts. The presentdisclosure includes such salts. Examples of applicable salt formsinclude hydrochlorides, hydrobromides, sulfates, methanesulfonates,nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g.(+)-tartrates, (−)-tartrates or mixtures thereof including racemicmixtures, succinates, benzoates and salts with amino acids, such asglutamic acid. These salts may be prepared by methods known to thoseskilled in art. Also included are base addition salts, such as sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present disclosure containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent or byion exchange. Examples of acceptable acid addition salts include thosederived from inorganic acids like hydrochloric, hydrobromic, nitric,carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived organicacids like acetic, propionic, isobutyric, maleic, malonic, benzoic,succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids, such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike. Certain specific compounds of the present disclosure contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents.

Certain compounds of the present disclosure can exist in unsolvatedforms as well as solvated forms, including hydrated forms. In general,the solvated forms are equivalent to unsolvated forms and areencompassed within the scope of the present disclosure. Certaincompounds of the present disclosure may exist in multiple crystalline oramorphous forms. In general, all physical forms are equivalent for theuses contemplated by the present disclosure and are intended to bewithin the scope of the present disclosure.

In addition to salt forms, the present disclosure provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentdisclosure. Additionally, prodrugs can be converted to the compounds ofthe present disclosure by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present disclosure when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

The term “wild type” refers to an organism which occurs in nature or maybe isolated from the environment and does not carry any geneticallyengineered mutations.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, quantities,characteristics, and other numerical values used in the specificationand claims, are to be understood as being modified in all instances bythe term “about” even though the term “about” may not expressly appearwith the value, amount or range. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are not and need not be exact, but maybe approximate and/or larger or smaller as desired, reflectingtolerances, conversion factors, rounding off, measurement error and thelike, and other factors known to those of skill in the art depending onthe desired properties sought to be obtained by the presently disclosedsubject matter. For example, the term “about,” when referring to a valuecan be meant to encompass variations of, in some embodiments, ±100% insome embodiments ±50%, in some embodiments ±20%, in some embodiments±10%, in some embodiments ±5%, in some embodiments ±1%, in someembodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

General Methods

a. Flow Cytometry

Single cell-suspensions were stained with antibodies after Fc blocking(BD bioscience). The following antibodies and staining reagents werepurchased from Biolegend: anti-CD45 (30-F11), anti-F4/80 (BM8),anti-CD11b (M1/70), Ly6C (HK1.4), Ly6G (RB6-8C5), WIC Class II(M5/114.15.2), CD90.1 (OX-7), CD8 (53-6.7), CD4 (RM4-5), Cell signaling:Calreticulin (D3E6), Thermofisher: LIVE/DEAD® Fixable Near-IR Dead CellStain Kit, TLR4 (UT41), CellROX™ Deep Red Flow Cytometry Assay Kit,Fixation and Permeabilization Buffer Set, and BD bioscience: TNF(MP6-XT22), GM-CSF(MP1-22E9), 7-AAD, BD, BD Cytofix/Cytoperm Plus Kit(with BD GolgiPlug) and staining were followed manufacture's protocol.Cells were acquired using BD FACSCalibur or BD FACSCelesta, and datawere analyzed using FlowJo (Tree Star).

b. Generation of BMDMs

For preparation of bone marrow cell suspensions, the bones of both hindlimbs (two tibias and two femurs) were flushed with ice-cold DMEMsupplemented with 10% FBS, 1% penicillin/streptomycin and 2 mML-glutamine (cell media) plus 20% L929-conditioned media. The cells wereincubated at 37° C., and on day 4, non-adherent cells were removed, andwashed with PBS. On day 7, BMDMs were lifted using Cellstripper(Mediatech, Manassas, Va.). BMDMs were seeded in 12-well plates, andtreated with 6-Diazo-5-oxo-L-norleucine (DON, Sigma Aldrich). To maketumor-conditioned media, tumor cells were cultured in the presence orabsence of DON (0.5 μM or 1 μM). After 1 hr of incubation, cells werewashed and replaced with drug-free fresh media. After 24 hours,supernatants were harvested and used as conditioned media (CM). BMDMwere cultured in the presence of these conditioned media for 24 hours.

c. Immunoblotting

For immunoblotting the nuclear and cytoplasm fractions, 10×10⁶ cellswere washed with PBS twice, then lysed cells in cytoplasm separationbuffer composed of 10 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.075% (v/v) NP40,1 mM DTT and 1 mM PMSF for cytoplasm fraction, and RIPA buffer with NaF,protease inhibitor, PMSF, sodium pyrophosphate, beta glycerophosphateand sodium vanadate for nuclear fraction. Western blotting was performedusing a standard protocol (Life Technologies). The following antibodieswere used from Cell Signaling: anti-active caspase 3, anti-p-STAT3(Tyr705), anti-STAT3, anti-p-NF-κB p65 (Ser536), anti-IDO, anti-LaminB,and anti-actin, and from abcam: anti-lamp2. All images were captured andanalyzed using UVP BioSpectrum 500 Imaging System.

Example 1

To explore the effect of DON on cancer, an EL4 mouse lymphoma model wasused and it showed that DON could markedly inhibit lymphoma growth,suggesting that bone marrow derived tumors may be exquisitelysusceptible to DON (FIG. 1). However, DON had a modest effect oninhibiting melanoma growth, which is a not a bone marrow derived tumor(FIG. 2).

Example 2

FIG. 3 shows that DON conditioned B16 melanoma to be killed byimmunotherapy by inhibiting tumor infiltrating regulatory T cells(Foxp3⁺).

Example 3 Summary

The glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON, 1) has shownrobust anti-cancer efficacy in preclinical and clinical studies, but itsdevelopment was halted due to marked systemic toxicities. Herein wedemonstrate that DON inhibits glutamine metabolism and providesantitumor efficacy in a murine model of gliobastoma, although toxicitywas observed. To enhance DON's therapeutic index, we utilized a prodrugstrategy to increase its brain delivery and limit systemic exposure.While these dual moiety prodrugs exhibited rapid metabolism in mouseplasma, several provided excellent stability in monkey and human plasma.The most stable compound (5c, methyl-POM DON-isopropyl-ester) wasevaluated in monkeys, where it achieved 10-fold enhanced brain:plasmaratio versus DON. This strategy may provide a path to DON utilization inGBM patients.

Introduction

Glioblastoma multiforme (GBM) is the most common and lethal form ofglioma (Ostrom, et al., 2015). Current therapeutic options that extendsurvival rates in GBM patients after tumor resection are limited toradiotherapy with concomitant administration of the DNA-alkylating agentTemodar™ and/or the carmustine releasing polymer Gliade™ (Weller, etal., 2014). But even under this standard of care, postoperative mediansurvival rates approximate 14.6 months, (Stupp, et al., 20015) andaverage five-year survival is less than 10% (Stupp, et al., 2009). Thereis, therefore, a significant unmet medical need for more effective GBMtreatments.

The ability of GBMs and other cancers rapidly and persistentlyproliferate often outgrowing vascular supplies necessitates that theydevelop a specialized metabolism (Schulze, et al., 2012; Hensley, etal., 2013). Glutamine metabolism plays an essential role in thisspecialized metabolism by entering the TCA cycle and then contributingto the biosynthetic pathways (nucleotide, protein and lipid synthesis)necessary for unchecked cell growth, (Hensley, et al., 2013) renderingcancer cells dependent on glutamine. This so called “glutamineaddiction” has been well characterized in GBMs (Dranoff, et al., 1985;Fogal, et al., 2015; Ru, et al., 2013; Tanaka, et al., 2015) and othercancers including leukemia/lymphomas, lung, triple-negative breastcancer and pancreatic cancer (Wise, et al., 2010; Hu, et al., 2009). Tocombat this glutamine addiction, several groups have explored theutility of selective glutaminase inhibitors (Erickson, et al., 2010;Lee, et al., 2014). Recently compounds that selectively targetglutaminase (GLS1) have been developed (McDermott, et al., 2016; Shukla,et al., 2012) and one such compound is in clinical trials (Konopleva, etal., 2015). However, the brain penetration of this GLS1 inhibitor ispoor (Gross, et al., 2014) and so far the clinical efficacy of thisapproach has been modest (Harding, et al., 2015).

Accumulating evidence shows that, as opposed to selective inhibition ofone glutamine utilizing enzyme, broadly antagonizing glutamineutilization is a highly effective means of inhibiting tumor cell growthin vitro and in vivo (Hensley, et al., 2013; Ahluwalia, et al., 1990;Dranoff, et al., Cancer Res, 1985). 6-Diazo-5-oxo-L-norleucine (DON),non-natural amino acid with structural similarity to glutamine, wasfirst isolated from Streptomyces bacteria in the early 1950's. Becauseof its reactive diazo group, DON has demonstrated the ability toalkylate several glutamine-utilizing enzymes such as glutaminase,(Thangavelu, et al., 2014) NAD synthase, (Barclay, et al., 1966) and CTPsynthetase (Hofer, et al., 2001) and FGAR aminotransferase (Grayzel, etal., 1960) in the purine and pyrimidine biosynthetic pathways,respectively. In preclinical models, DON robustly inhibited the growthof glutamine-dependent human cancer cells in vitro, and reduced tumorsize and improved survival rates in vivo (Ahluwalia, et al., 1990;Cervantes-Madrid, et al., 2015). Because of the robust preclinical data,DON was evaluated in several clinical studies where it demonstratedpromising results (Eagan, et al., 1982; Earhart, et al., 1982; Lynch, etal., 1982; Magill, et al., 1957; Rahman, et al., 1985; Sklaroff, et al.,1980; Sullivan, et al., 1962). For example, DON administrationcaused >50% regression or stable disease in late stage adult patients(Magill, et al., 1957) and in children with hematological malignanciesor solid tumors (Sullivan, et al., 1988). Unfortunately, its developmentwas hampered by dose limiting toxicities, many of which were GI-related(Magill, et al., 1957; Rahman, et al., 1985; Earhart, et al., 1990) asthe GI system is highly dependent on glutamine utilization.

One strategy to improve the therapeutic index of DON for GBM therapywould be to increase its brain exposure while limiting its systemicexposure and thus toxicity (Upadhyay, et al., 2014). The prodrugapproach is a well-established strategy to alter the pharmacokinetic andtissue distribution of drug molecules, however synthetically thisapproach is challenging with DON. Applicant has found that compoundshaving formula (I), formula (IIA), formula (IIB), and formula (III)exhibit unexpected enhanced CSF to plasma partitioning afteradministration, making them uniquely useful for the treatment of CNScancers such as glioblastoma.

Herein we describe the efficacy of DON in a murine model of GBM,although overt toxicities were observed. Several DON prodrugs weresynthesized using three types of amine promoieties including(oxodioxolenyl)methyl carbamate esters (, dipeptides, andpivaloyl-oxy-methyl (POM)-based esters. The dual promoiety-containingprodrugs resulted in sufficient chemical stability permitting furtherevaluation in in vitro metabolic stability assays. While all of theprodrugs exhibited rapid metabolism in mouse plasma, some providedsurprising plasma stability in monkeys and humans. When evaluated invivo, the most stable DON prodrug (5c, methyl-POM-DON-isopropyl-ester)achieved an unexpected 10-fold enhanced brain: plasma ratio versus DONin monkeys, thus providing a possible clinical path to DON utilizationin GBM patients.

Results and Discussion DON Showed Robust Inhibition of GlutamineMetabolism and Antitumor Efficacy in a Murine GBM Model

Despite several lines of evidence indicating the potential therapeuticefficacy of targeting glutamine metabolism in GBM, the effect of DON onGBM tumor growth has not yet been reported in vivo. Using the U87 flankxenograft mouse model of GBM, (Eshleman, et al., 2002) we firstconfirmed that systemic administration of DON (0.8 mg/kg, i.p) inhibitedglutamine metabolism as reflected by an accumulation of endogenousglutamine in the tumor (FIG. 5A; p<0.05) similar to other model systems.(Willis, et al., 1977; Windmueller, et al., 1974) We next evaluated itsantitumor efficacy, and observed that DON not only halted tumor growth,but also effectively induced tumor regression. Specifically,vehicle-treated mice displayed significant tumor growth over the courseof the experiment, while DON-treated mice (0.8 mg/kg, i.p, q.d.)exhibited >50% reduction in tumor volume (FIG. 5B; main effect of time[F(3,48)=6.049, p=0.0014]; treatment [F(1,16)=33.42, p<0.0001];interaction [F(3,48)=21.70, p<0.0001]). Although DON exhibited excellentanti-tumor efficacy, all mice receiving DON displayed significant signsof toxicity including weight loss (12±4.1%), hunching, ptosis, andlethargy. These findings are consistent with other reports of DON'sefficacy and toxicity both in vitro and in vivo. (Fogal et al., 2015;Cervantes-Madrid, et al., 2015; Potter, et al., 2015)

Simple DON Alkyl Ester Prodrugs Found to be Unstable. Masking Both DON'sCarboxylate and Amine Functionalities Required to Obtain StableProdrugs.

A prodrug strategy is often employed to enhance tissue penetration andchange the pharmacokinetic parameters of effective drugs. Indeed,prodrug strategies are common in drug development as 5-7% of theapproved worldwide drugs are prodrugs. (Rautio, et al., 2008) Ourinitial prodrug strategy for DON involved masking the carboxylic acidwith simple alkyl esters such as ethyl 2a and isopropyl 2b. Thesynthesis of these two derivatives was straightforward affordingcompounds 2a and 2b in good yield. It is surprising to us that thesesimple DON alkyl esters had not previously been reported in the chemicalliterature, given that DON chemistry and utility has been described bynumerous groups for over 60 years. (Magill, et al., 1957; Dion, et al.,1956; Magill, et al., 1956; Coffey, et al., 1956) One potential reasonis that we discovered that 2a and 2b were unstable, slowing cyclizing toform unique diazo-imines 9a and 9b. These two unique derivatives werefound to be chemically stable even at acidic pH, precluding their use asDON prodrugs.

Given the instability of simple ester prodrugs, we next masked both theprimary amine and the carboxylate of DON with prodrug moieties. Thisdual promoiety strategy was rationalized to eliminate the potential forcyclization and potentially further improve the lipophilicity. Weutilized three amine promoieties including (oxodioxolenyl)methylcarbamate esters, dipeptides, and pivaloyl-oxy-methyl (POM)-basedesters. These promoeities were chosen because they target distinctmetabolic enzymes including paraoxonase, aminopeptidases, andcarboxylesterases, respectively. To impart further metabolic stabilityof the POM derivative, we prepared corresponding methyl-POM analogs. Alldual promoiety-containing prodrugs exhibited sufficient chemicalstability to permit further evaluation.

All DON Prodrugs were Rapidly Metabolized in Mouse Plasma, However 5band 5c Found to be Stable in Human and Monkey Plasma

Table 2 outlines the plasma stability of representative DON. Allprodrugs were completely metabolized in mouse plasma within the 60 minincubation time. However in monkey and human plasma, the prodrugs 5b and5c, with methyl-POM on the amine and ethyl or isopropyl ester on thecarboxylate respectively, demonstrated moderate/high stability with60-75% of the prodrug remaining in monkey plasma, and 80-90% remainingin human plasma within the 60 min incubation time. Given that compound5c (also referred to as compound 14b, see Table 1) had the beststability profile in human plasma, it was selected for furtherevaluation in pharmacokinetic studies and compared to DON for itsability to penetrate the brain and liberate DON.

TABLE 2 Plasma stability of DON prodrugs following 60 min incubation inmouse, monkey and human plasma. Compound PLASMA STABILITY # Mouse MonkeyHuman 36 0 0 0 13 0 0 0 25 0 1 1  9 0 1 1 34 0 4 12 38 0 10 30 42 0 0 932 0 75 88 5c or 14b 0 61 91

Prodrug 5c Enhanced Brain Delivery of DON in Monkeys but not in Mice

As expected from a DON prodrug which is completely metabolized in mouseplasma, we found that oral administration of DON (1) (0.8 mg/kg) and 5c(0.8 mg/kg equivalent) exhibited similar DON plasma (FIG. 6) and brain(FIG. 6) concentration profiles when dosed in mice. The AUC_(0-t) of DONfollowing administration of DON and 5c in plasma were 1.25 nmol*h/mL and1.22 nmol*h/mL respectively, suggesting rapid and complete liberation ofDON from 5c in vivo. Similarly in the mouse brain, the AUC_(0-t) of DONfollowing DON or 5c administration was 0.57 nmol*h/g and 0.69 nmol*h/g,respectively, with the brain/plasma approximately 0.46 from DON vs 0.56from prodrug 5c. These pharmacokinetic results corroborated the in vitrometabolism studies suggesting 5c was completely converted to DON inmouse plasma.

Following the mouse studies, we evaluated the pharmacokinetics of DONand 5c in monkeys, as monkeys better mimicked the human plasma stabilityprofile. In pigtail macaques, i.v. administration of DON and 5c (1.6mg/kg DON equivalent) demonstrated significantly different DON plasmaprofiles (FIG. 7). DON administration provided high plasma exposureswith AUC_(0-t) of 42.7 nmol*h/mL. In contrast, 5c administrationdelivered ˜7 fold lower plasma exposure of DON with AUC_(0-t) of 5.71nmol*h/mL. The opposite observation was seen in the CSF where enhancedDON levels were observed after 5c administration. In the CSF at 30 minpost dose, DON administration resulted in 0.33 nmol/g DON while 5cdelivered 1.43 nmol/g DON. When comparing plasma to CSF ratio at 30 min,5c demonstrated 10-fold enhancement of DON CSF delivery versus DON (FIG.7A-C).

Conclusion

We demonstrate efficacy of DON in a murine model of GBM, although overttoxicity was observed. To enhance DON's therapeutic index, we utilized aprodrug strategy to increase its brain delivery and limit its systemicexposure. While our dual promoeity prodrugs exhibited rapid metabolismin mice, our lead prodrug 5c provided excellent stability in human andmonkey plasma, and achieved a 10-fold enhanced brain:plasma ratio versusDON in monkeys. This strategy may provide a path to DON utilization inGBM patients.

Mice Efficacy Studies

All mouse efficacy studies were conducted according to protocol #M013M69approved by the Animal Care and Use Committee at Johns HopkinsUniversity. Female athymic (RH_Foxn1nu mice) mice between 25 and 30 gwere obtained (Harlan Sprague Dawley Inc, Indianapolis, Ind.), andmaintained on a 12 hour light-dark cycle with ad libitum access to foodand water. U87 human glioma cells were injected s.c. (5×10⁶ cells in 100ml of PBS) in four separate locations on the flanks of each mouse. Whentumors grew to a mean volume of around 200 mm³, mice were randomizedinto either vehicle (HEPES-buffered saline, i.p.) or DON (1; 0.8 mg/kg,i.p.). In one cohort, mice were administered a single dose of theappropriate solution two hours after which glutamine levels werequantified in the tumor as described previously.⁴⁹ In brief, tumors wereharvested, snap frozen, and homogenized in liquid N2 then subjected tometabolite extraction using methanol and DI water. Quantification wasperformed using Agilent 6520 Quadrupole-Time-of-Flight (Q-TOF) massspectrometer with Agilent 1290 HPLC and using Agilent Mass Hunter andAgilent Qualitative and Quantitative Analysis Software packages.Glutamine content was averaged by group for each individual tumor(n=3-4/group), depicted as relative intensity, and analyzed byone-tailed t test. In a second cohort, efficacy experiments wereconducted. Mice were injected once daily for six days; tumor volumeswere measured using digital calipers and calculated according to theformula: [volume=(largest tumor dimension)×(smallest tumordimension)×0.52] at 2, 4, and 6 days after the onset of treatment. Eachindividual tumor (n=8-10/group) was normalized to its pretreatmentvolume, averaged and analyzed by repeated measures two-way analysis ofvariance (ANOVA). If significant, a Bonferroni post hoc test wassubsequently applied. Significance was defined as p<0.05.

In Vitro Metabolic Stability Studies

For metabolic stability, plasma from rodent (mouse) and non-rodentspecies (human and monkeys) were used. For stability, prodrugs (10 μM)were spiked in each plasma matrix and incubated in an orbital shaker at37° C. At predetermined times (0 and 60 min), 100 μL aliquots of themixture in duplicate were removed and the reaction quenched by additionof three times the volume of ice cold acetonitrile spiked with theinternal standard (losartan 5 μM). The samples were vortexed for 30 sand centrifuged at 12000 g for 10 min. 50 μL of the supernatant wasdiluted with 50 μL water and transferred to a 250 μL polypropylene vialsealed with a Teflon cap. Prodrug disappearance was monitored over timeusing a liquid chromatography and tandem mass spectrometry (LC/MS/MS)method as described below.

Pharmacokinetic Studies in Mice

All pharmacokinetic studies in mice were conducted according to protocol(#M013M113) approved by the Animal Care and Use Committee at JohnsHopkins University. C57BL/6 mice between 25 and 30 g were obtained fromHarlan, and maintained on a 12 hour light-dark cycle with ad libitumaccess to food and water. To evaluate the brain and plasmapharmacokinetics of DON and its prodrug 5c, 8-12 week old C57BL/6 wereadministered DON (1; 0.8 mg/kg, p.o. in phosphate-buffered saline) andits prodrug 5c (at 0.8 mg/kg equivalent DON (1), p.o. inphosphate-buffered saline with 5% EtOH and 5% Tween-80). The mice weresacrificed by pentobarbital injection at 10, 30 and 90 minutes post drugadministration, and blood was collected via cardiac puncture and placedinto iced EDTA coated BD microtainers. Blood samples were spun at 2,000g for 15 minutes, and plasma was removed and stored at −80° C. Braintissues were harvested following blood collection and immediately snapfrozen in liquid nitrogen and stored at −80° C. until LC/MS analysis.

Pharmacokinetic Studies in Non-Human Primates

All monkey studies were conducted according to protocol (#PR15M298)approved by the Animal Care and Use Committee at Johns HopkinsUniversity. Two female pigtail monkeys (approximately 3.5 kg, non-drugnaive) were adjacently housed in stainless steel cages on a socialinteraction rack (contains 4 cages, each 32.5″ wide×28″ deep×32″ high)maintaining temperature of 64-84° F., humidity of 30-70% withalternating 14-10 hour light/dark cycle as per the USDA Animal WelfareAct (9 CFR, Parts 1, 2, and 3). Food was provided daily in amountsappropriate for the size and age of the animals and RO purified waterprovided ad libitum through an in-cage lixit valve. Food enrichment wasprovided Monday through Friday. Prior to drug administration, macaqueswere sedated with ketamine given as an intramuscular injection prior totest article administration. Sedation was maintained through blood andcerebrospinal fluid (CSF) sample collections with ketamine at a startingrate of 15 mg/kg with additional doses of 20-30 mg during the firsthour. At subsequent time points ketamine was given at 10-15 mg/kg. DON(50 mM HEPES buffered saline) and 5c (Diastereoisomer 1), (50 mM HEPESbuffered saline containing 5% ethanol and 5% tween) were administered(1.6 mg/kg equivalent) to the animals at a dosing volume of 1 mL/kgintravenously. CSF sample (target of 50 μL) was obtained by percutaneouspuncture of the cisterna magna at 30 min post dose. Blood samples (1 mL)were collected at 15, 30, 1, 2 4 and 6 h post dose by percutaneouspuncture of a peripheral vein. Samples were processed for plasma(centrifuged at a temperature of 4° C., at 3,000×g, for 10 minutes). Allsamples were maintained chilled on ice throughout processing. Sampleswere collected in microcentrifuge tubes, flash frozen, and placed in afreezer set to maintain −80° C. until LC/MS analysis.

Bioanalysis of DON

We have previously published a highly sensitive method for analysis ofDON in biological matrices (Alt, et al., 2015). However due to chemicallability of DON and its prodrugs, a milder derivatization methodemploying dabsyl chloride was developed and validated. Briefly, DON wasextracted from samples (50 mg) with 250 μL methanol containingGlutamate-d5 (10 μM ISTD) by vortexing in low retention tubes. Sampleswere centrifuged at 16,000×g for 5 minutes to precipitate proteins.Supernatants (200 μL) were moved to new tube and dried at 45° C. undervacuum for 1 hour. To each tube, 50 μL of 0.2 M sodium bicarbonatebuffer (pH 9.0) and 100 μL of 10 mM dabsyl chloride in acetone wasadded. After vortexing, samples were incubated at 60° C. for 15 minutesto derivatize. Samples (2 μL) were injected and separated on an Agilent1290 equipped with a an Agilent Eclipse plus C18 RRHD 2.1×100 mm columnover a 2.5 minute gradient from 20-95% acetonitrile+0.1% formic acid andquantified on an Agilent 6520 QTOF mass spectrometer. Calibration curvesover the range of 0.005-17.1 μg/mL in plasma and CSF for DON wereconstructed from the peak area ratio of the analyte to the internalstandard using linear regression with a weighting factor of 1/(nominalconcentration). Correlation coefficient of greater than 0.99 wasobtained in all analytical runs. The mean predicted relative standarddeviation for back calculated concentrations of the standards and QC'sfor all analytes were within the range of 85 to 115%, except for thelowest concentration which was within the range of 80 to 120% with anoverall accuracy and precision of 6.7% and 6.6% respectively.

Pharmacokinetic Analysis

Mean concentration-time data was used for pharmacokinetic analysis.Non-compartmental-analysis module in WinNonlin® (version 5.3) was usedto assess pharmacokinetic parameters. Peak plasma concentrations(C_(max)) and time to C_(max) (T_(max)) were the observed values. Areaunder the curve (AUC) was calculated by log-linear trapezoidal rule tothe end of sample collection (AUC_(last)).

Example 4

In head-to-head comparisions, 25 was found to be markedly andunexpectedly more effective than a clinical stage selective glutaminaseinhibitor CB-839.

FIG. 8 illustrates that 25 (5 day dosing starting on day 7) is superiorto CB-839 (30 day dosing starting day 1) in a CT26 tumor model.

FIG. 9 illustrates that 25 (4 days starting on day 6) is superior toCB-839 (continuous twice daily dosing starting on day 1 prior toengraftment) in a CT26 tumor model. Mice received daily 25 (1.9 mg/kg)on days 6-9 as compared to BID glutaminase inhibitor on days 1-15.

FIG. 10 illustrates that 25 (daily days 7-22) is superior to CB-839(continuous twice daily dosing days 1-29) in a 4T1 breast cancer model.Mice received daily 25 (1.0 mg·kg/d) for days 7-22 as compared to BIDglutaminase inhibitor for days 1-29.

Example 5

DON pro-drugs demonstrate efficacy in multiple tumor types, includingefficacy in B & T cell lymphomas, colon cancers, breast cancer andmelanoma. Daily dosing provides effective monotherapy. Every other daydosing leads to minimal resistance. FIG. 11 illustrates that 25 dosingof 1 mg/kg following by 0.3 mg/kg leads to a complete and durableresponse in the MC38 tumor.

FIG. 12 illustrates that 25 providese a robust response and improvedoverall survival in multiple tumor models, including CT26 Colon Cancer.

FIG. 13 illustrates that 25 provides a robust response and improvedoverall survival in multiple tumor models, including 4T1 Breast Cancer.

FIG. 14 illustrates that mice cured with 25 alone immunologically rejecttumors upon re-challenge, demonstrating that monotherapy with certainDON prodrugs, such as 25 monotherapy, is immunotherapy.

FIG. 15 additionally illustrates that 25 is immunotherapy.

Example 6 DON/DON Prodrugs Condition Tumors to Immunotherapy andSignificantly Enhance the Response to Checkpoint Inhibitors, AdoptiveCell Transfer and A2aR Inhibition

Conclusions: DON/DON prodrugs robustly enhance the immune mediatedresponse to therapy with anti-PD1, the response to adoptive celltransfer, and the response to adenosine A2a receptor blockade.Immunotherapy fails patients with rapidly progressive disease due to thelag in response. 30-40% of patients treated with anti-PD1 therapyprogress rapidly in the first few months, chemotherapy showed an earlysurvival advantage in NSCLC likely due to it's quick effect on rapidlygrowing disease compared to immunotherapy, and conditioning and adjuvanttherapies to immunotherapy must be able to control tumor growth to beeffective in these patients.

FIG. 16 illustrates that glutamine inhibition (e.g., using DON) reducesthe oxygen consumption and lactate production of tumor cells. FIG. 17illustrates that glutamine inhibition (e.g., using DON) also improvedthe CD8/Treg ratio in the tumor and reduces hypoxia in the TILs.

FIG. 18 illustrates that 25 conditions tumors to be eliminated byanti-PD1 therapy in the MC38 Model, and that 25 rescues anti-PD1failures. In addition, FIG. 19 illustrates that even in the moredifficult CT26 model, 25 unexpectedly enhances the response to anti-PD1.Similarly, FIG. 20 illustrates that inhibiting glutamine metabolismunexpectedly potentiates the anti-tumor response to A2aR inhibition.Finally, FIG. 21 illustrates that inhibiting glutamine metabolismunexpectedly enhances the efficacy of adoptive cellular therapy (ACT) ina B16-OVA model.

Example 7 Compound 14b Enhanced CSF Delivery of DON in Monkey Method

Compound: Compound 14b was dissolved in 50 mM HEPES buffered salinecontaining 5% ethanol and 5% tween on the date of administration.

Monkey: Monkey studies were conducted according to protocol (#PR15M298)approved by the Animal Care and Use Committee at Johns HopkinsUniversity. Two female pigtail monkeys (approximately 3.5 kg, non-drugnaive) were adjacently housed in stainless steel cages on a socialinteraction rack (contains 4 cages, each 32.5″ wide×28″ deep×32″ high)maintaining temperature of 64-84° F., humidity of 30-70% withalternating 14-10 hour light/dark cycle as per the USDA Animal WelfareAct (9 CFR, Parts 1, 2, and 3). Food was provided daily in amountsappropriate for the size and age of the animals and RO purified waterprovided ad libitum through an in-cage lixit valve. Food enrichment wasprovided Monday through Friday.

Treatment: Prior to drug administration, macaques were sedated withketamine given as an intramuscular injection prior to test articleadministration. Sedation was maintained through blood and cerebrospinalfluid (CSF) sample collections with ketamine at a starting rate of 15mg/kg with additional doses of 20-30 mg during the first hour. Atsubsequent time points ketamine was given at 10-15 mg/kg. DON (50 mMHEPES buffered saline) and compound 14b (50 mM HEPES buffered salinecontaining 5% ethanol and 5% tween) were administered (1.6 and 3.6 mg/kgequivalent dose of DON) to the animals at a dosing volume of 1 mL/kgintravenously. CSF sample (target of 50 μL) was obtained by percutaneouspuncture of the cisterna magna at 30 min post dose. Blood samples (1 mL)were collected at 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h post dose bypercutaneous puncture of a peripheral vein. Samples were processed forplasma (centrifuged at a temperature of 4° C., at 3,000 g, for 10minutes). All samples were maintained chilled on ice throughoutprocessing. Samples were collected in microcentrifuge tubes, flashfrozen, and placed in a freezer set to maintain −80° C. until LC/MSanalysis.

Data Analysis: DON was extracted from samples (50 mg) with 250 μLmethanol containing glutamate-d5 (10 μM ISTD) by vortexing in lowretention tubes. Samples were centrifuged at 16,000 g for 5 minutes toprecipitate proteins. Supernatants (200 μL) were moved to new tubes anddried at 45° C. under vacuum for 1 hour. To each tube, 50 μL of 0.2 Msodium bicarbonate buffer (pH 9.0) and 100 μL of 10 mM dabsyl chloridein acetone was added. After vortexing, samples were incubated at 60° C.for 15 minutes to derivatize. Samples (2 μL) were injected and separatedon an Agilent 1290 equipped with an Agilent Eclipse plus C18 RRHD2.1×100 mm column over a 2.5 minute gradient from 20-95%acetonitrile+0.1% formic acid and quantified on an Agilent 6520 QTOFmass spectrometer. Calibration curves over the range of 0.005-17.1 μg/mLin plasma and CSF for DON were constructed from the peak area ratio ofthe analyte to the internal standard using linear regression with aweighting factor of 1/(nominal concentration). Correlation coefficientof greater than 0.99 was obtained in all analytical runs. The meanpredicted relative standard deviation for back calculated concentrationsof the standards and QC's for all analytes were within the range of 85to 115%, except for the lowest concentration which was within the rangeof 80 to 120% with an overall accuracy and precision of 6.7% and 6.6%respectively.

Results

The pharmacokinetics of DON and compound 14b in monkeys were evaluated.In pigtail macaques, i.v. administration of DON (1.6 mg/kg) and compound14b (3.6 mg/kg; 1.6 mg/kg DON equivalent) demonstrated significantlydifferent DON plasma profiles (FIG. 38A). DON administration providedhigh plasma exposures with AUC0-t of 42.7 nmol*h/mL. In contrast,compound 14b administration delivered ˜7 fold lower plasma exposure ofDON with AUC0-t of 5.71 nmol*h/mL. The opposite observation was seen inthe CSF where enhanced DON levels were observed after compound 14badministration. In the CSF at 30 min post dose, DON administrationresulted in 0.33 nmol/g DON while compound 14b delivered 1.43 nmol/gDON. When comparing plasma to CSF ratio at 30 min, compound 14bdemonstrated unexpected 10-fold enhancement of DON CSF delivery versusDON (FIG. 38B).

Example 8 Compounds 14b and 47 Enhanced CSF Delivery of DON in SwineMethod

Compound: Compound 47 was dissolved in a sterile saline containing 5%ethanol and 5% Tween 80 on the date of administration.

Swine: Swine studies were conducted under a protocol approved by theJohns Hopkins Animal Care and Use Committee. Adult, femaleGottingen×Yucutan miniature swine (Massachusetts General Hospital, MA)were housed in Johns Hopkins University facilities accredited by theAssociation for Assessment and Accreditation of Laboratory Animal CareInternational in compliance with the Animal Welfare Act, Animal WelfareRegulations, and the Public Health Service Policy on the Humane Care andUse of Laboratory Animals. Animals were maintained on a 14-h light and10-h dark schedule, provided ad libitum water and a commercial miniswinediet (Teklad, Madison, Wis.) with environmental enrichment(fruit/vegetables) twice daily.

DON and Compounds 14b and 47 Treatment: Animals were individually housedwhile on study in order to monitor behavior and clinical healthfollowing drug administration. Whole blood for drug pharmacokineticevaluation was collected from a dual lumen central venous catheter (CVC)implanted in the external jugular vein prior to study initiation.Animals were anesthetized with a combination of ketamine hydrochloride(20-30 mg/kg, i.m.) and xylazine (2 mg/kg, i.m.), intubated, andmaintained under isoflurane (1-2%) inhalant anesthesia. A temporaryperipheral saphenous vein catheter was placed in the hind limb to allowfor anatomical separation of drug infusion and whole blood sampling viaCVC. DON and compounds 14b and 47 were dissolved in a sterile salinesolution containing 5% ethanol and 5% Tween 80 prior to i.v. infusionvia saphenous vein catheter over 1 hour (1 ml/min) for a final dose of1.6 mg/kg or molar equivalent administered at 1 ml/kg (n=1/dose). Bloodsamples (1 mL) were taken from CVC at predose, 5, 15, 30, 45, and 60min. Plasma was separated by low speed centrifugation at 3000 g for 10min at 4° C. CSF was obtained from the cisterna magna using a 3.5 in×22gauge spinal needle (Becton Dickinson Health Care, Franklin Lakes, N.J.,USA) at 60 min post-dose. All samples were flash frozen upon harvest andstored at −80 C until bioanalysis.

Data analysis: Quantitation of DON in plasma, CSF, and brain homogenateby LC-MS/MS was performed. Briefly, DON was extracted from plasma, CSF,and brain samples with methanol containing glutamate-d5 (10 μM ISTD) byvortexing followed by centrifugation 16000 g for 5 min. Supernatantswere aliquoted and dried at 45° C. for under vacuum for 1 h. Sodiumbicarbonate buffer (0.2M, pH 9.0) and dabsyl chloride (10 mM) in acetonewere added to each tube, mixed, and incubated for 15 min at 60° C. toderivatize. Samples were then injected and separated on an Agilent 1290equipped with an Agilent Eclipse plus C18 RRHD 2.1×100 mm column over a2.5 min gradient from 20 to 95% acetonitrile+0.1% formic acid andquantified on an Agilent 6520 QTOF mass spectrometer. Peak area ratio ofthe analyte to the internal standard was plotted against a 14 standardcurve to yield DON concentrations for each sample.

Result

The pharmacokinetic of DON, compound 14b and compound 47 were evaluatedin swine. IV administration of compounds 14b and 47 (1.6 mg/kg DONequivalent dose) resulted in 3-5-fold lower DON plasma exposuresrelative to an equimolar dose of DON (FIG. 39A). Plasma AUC_(0-t) forDON and compounds 14b and 47 were 29.9, 8.00 and 5.70 nmol·h/mL,respectively. The opposite trend occurred in CSF, where compounds 14band 47 delivered substantially higher amounts of DON to the CSF (FIG.39B), resulting in unexpected increased CSF-to-plasma ratios (FIG. 39C).

Example 9 Compound 25 Enhances Immunotherapy in EO771 Tumor-Bearing MiceMethod

Mouse: C57BL/6 (both male and female mice, 6-8 weeks of age) werepurchased from Jackson Laboratories. The studies were conducted under aprotocol approved by the Johns Hopkins Animal Care and Use Committee.

Cell Line: EO771 breast cancer cell lines were purchased from CH3BioSystems. EO771 cells were cultured in RPMI supplemented with 10% FBS,1% penicillin/streptomycin. They were regularly tested to confirmmycoplasma free using MycoAlert mycoplasma detection kit (Lonza). Cellswere never passaged more than 3 weeks before use in experiment. EO771cells (2×10⁵ cells in 200 μl per mouse) were subcutaneously inoculatedinto the mammary fat pad (C57BL/6).

Treatment with compound 25: EO771 tumor-bearing mice were treated withglutamine antagonist prodrug, compound 25 (1 mg/kg) starting at day 7after tumor inoculation. After 7 days, lower dose (0.3 mg/kg) ofglutamine antagonist prodrug was applied.

Treatment with immunotherapy alone or combined immunotherapy withcompound 25: On days 9, 12, and 15, mice were injected with or without100 μg anti-PD1 alone or 100 μg anti-PD1 in combination with 100 μganti-CTLA4, followed by treatment with or without compound 25. Eachindividual mouse tumor growth and survival curves were recorded.

Result: Treatment with compound 25 alone resulted in delayed tumorgrowth and increased survival (FIGS. 40A, B, and E). Anti-PD-1 therapyalone resulted in delayed tumor growth (FIGS. 40C and D). Thecombination of compound 25 and anti-PD-1 therapy resulted in asurprising increase in inhibition of tumor growth and survival (FIGS.40F and G). Taken together, these data show that combining metabolictherapy with checkpoint blockade leads to a unexpected increase insusceptibility of the tumor to immunotherapy.

Example 10 Compound 25 Enhances Immunotherapy in 4T1 Tumor-Bearing Mice

Mouse: BALB/cJ (both male and female mice, 6-8 weeks of age) werepurchased from Jackson Laboratories. The studies were conducted under aprotocol approved by the Johns Hopkins Animal Care and Use Committee.

Cell Line and tumor model: 4T1 breast cancer cell lines were purchasedfrom the ATCC. 4T1 cells were cultured in RPMI supplemented with 10%FBS, 1% penicillin/streptomycin. They were regularly tested to confirmmycoplasma free using MycoAlert mycoplasma detection kit (Lonza). Cellswere never passaged more than 3 weeks before use in experiment. 4T1cells (1×10⁵ cells in 200 μl per mouse) were subcutaneously inoculatedinto the mammary fat pad. (BALB/cJ).

Treatment with immunotherapy alone: 0.1×10⁶ 4 T1 cells were inoculatedsubcutaneously into mammary fat pad in BALB/cJ female mice. On day 7,10, 13, 17, and 24, mice were injected IP with 250 μg anti-PD1 and/oranti-CTLA4 antibodies. Tumor size was monitored. FIGS. 41A-G show that4T1 tumor cells are resistant to immunotherapy in the form of checkpointblockade.

Treatment wtih compound 25, immunotherapy, or combination of compound 25and immunotherapies: Mice were injected with 4T1 tumors as describedabove and treated on day 7 post injection with either no treatment (NT),compound 25 alone, anti-PD-1+anti-CTLA-4, or compound25+anti-PD1+anti-CTLA-4. FIGS. 41H and I show that mice treated withanti-CTLA4 and anti-PD1 showed minimal therapeutic benefit compared tothe vehicle treated group. FIG. 41J shows that the compound 25 treatedgroup displayed a modest decrease in tumor growth. FIGS. 41K and L showthat when mice were treated with the combination compound 25, anti-PD1,and anti-CTLA4, a substantial decrease of tumor growth and increase insurvival time were observed. These findings demonstrate that alteringTME glutamine inhibition unexpectedly enhances the efficacy ofcheckpoint blockade in tumors that are resistant to such therapy.

Example 11 Compound 25 Reduced Spontaneous Lung Metastasis in 4T1Tumor-Bearing Mice

Treatment with compound 25: 0.1×10⁶ 4 T1 cells were implantedsubcutaneously into mammary fat pad in BALB/cJ female mice. 4T1tumor-bearing mice were treated with compound 25 (1 mg/kg) starting atday 7 after tumor inoculation. After 7 days, lower dose (0.3 mg/kg) ofcompound 25 was used. On day 30, the whole lung were harvested, andspontaneous lung metastases were analyzed by inflation with 15% indiaink, to quantify tumor nodules, or by flow cytometry (FIG. 42A). FIG.42B quantifies spontaneous lung metasteses in lungs of mice with notreatment (NT) and treatment with compound 25. These findingsdemonstrate that compound 25 significantly reduces lung metastasis.

Example 12 Compound 25 Inhibited Tumor Growth in Syngeneic Mouse Models

Tumor growth and survival experiments: Tumor injections wereadministered in the right flank. For the MC38 and MC38OVA models,C57BL/6 WT mice were injected with 5×10⁵ MC38 or MC38OVA cells (s.c.)cultured in DMEM-based media. For the CT26 model, BALB/c mice wereinjected with 5×10⁵ CT26 cells (s.c.) cultured in RPMI-based media.Compound 25 was dissolved in 2.5% ethanol in PBS (v/v) which wasadministered for all vehicle-treated control experiments. For MC38 andMC38OVA experiments, mice were treated with Compound 25 or vehicle bydaily gavage with 1 mg/kg/day in 100 uL for days 10-14 and with 0.3mg/kg/day for day 15-24. For CT26 experiments, mice were treated withcompound 25 or vehicle by daily gavage with 1 mg/kg/day in 100 uL fordays 7-11 and with 0.3 mg/kg/day for day 12-21.

Results: Mice were injected subcutaneously with MC38 colon cancer, EL-4lymphoma, CT26 colon cancer, and B16 melanoma. In each case treatmentwith compound 25 led to a marked decrease in tumor growth. FIGS. 43A-Cshow tumor growth and survival over time of MC38-bearing mice treatedwith vehicle and compound 25. In the case of MC38, monotherapy treatmentwith compound 25 for 14 days led to durable cures. FIGS. 44A-B showtumor growth and survival over time of CT26 tumor-bearing mice treatedwith vehicle and compound 25. FIGS. 45A-B show tumor growth and survivalover time of B16 tumor-bearing mice treated with vehicle and compound25. FIGS. 46A-B shows tumor growth and survival over time of EL4tumor-bearing mice treated with vehicle and compound 25.

Example 13 Compound 25 Enhanced the Efficacy of Immunotherapy in MC38and CT26 Bearing Mice

Treating MC38 tumor-bearing mice: MC38 bearing C57BL/6 mice were treatedwith vehicle, anti-PD-1, compound 25, or the combination of compound 25and anti-PD-1 beginning on day 10 after tumor inoculation. The micetreated with compound 25 and anti-PD-1 showed an unexpected increase inboth tumor regression and complete responses compared with anti-PD-1therapy alone. See FIGS. 47A-F.

Treating CT26 tumor-bearing mice: CT26 bearing BALB/c mice were treatedwith vehicle, anti-PD-1, compound 25, or combination of compound 25 andanti-PD-1 beginning on day 7 after tumor inoculation. Combinationtherapy produced significant anti-tumor responses in the CT26 model. SeeFIGS. 48A-F.

Example 14 Compound 25 Enhanced Endogenous Anti-Tumor Immunity

Mice that had been cured by monotherapy were rechallenged with an equalburden of tumor injected on the opposite flank. More than three-quartersof mice cured by treatment with compound 25 as a single agentunexpectedly rejected MC38 rechallenge. See FIG. 49A.

To confirm the immunologic basis of this phenomenon, MC38-bearingRAG2−/− mice and wild type mice treated with compound 25 were compared.While glutamine blockade with compound 25 had some initial effect ontumor growth in the RAG2−/− mice, tumor growth rate recovered afterseveral days (FIG. 49C). In contrast, treatment for 14 days of WT mice(with intact adaptive immune responses) led to significant control ofthe tumor including complete regression in a proportion of the mice(FIGS. 49B and D). These results demonstrate that glutamine blockadewith compound 25 produces a surprising enhancement in endogenousanti-tumor immunity.

Example 15 Compound 25 Enhanced the Efficacy of Adoptive CellularTherapy

Adoptive Cellular Therapy: OVA-expressing B16 melanoma model, C57BL/6 WTmice received a s.c. injection of 2×10⁵ B16-OVA melanoma cells culturedunder OVA selection media containing 400 μg/ml G418 (Life technologies).Mice were treated with compound 25 or vehicle by daily gavage with 1mg/kg/day in 100 uL for days 7-9 post tumor inoculation. Ten days aftertumor injection, mice received an adoptive transfer of 1.5×10⁶ activatedOT1 cells, which had been stimulated in vitro with SIINFEKL peptide for48 h, expanded in IL-2 (1 ng/mL) for 24 h and isolated with Ficolgradient centrifugation. Mice were randomized based on tumor size beforetransfer of activated OT1 cells for adoptive transfer experiments. Tumorburden was assessed every 2-4 days by measuring length and width oftumor. Tumor volume was calculated using the formula V=(L×W×W)/2, whereV is tumor volume, W is tumor width, and L is tumor length. Mice weresacrificed when tumor reached 2 cm in any dimension, became ulcerate ornecrotic, or caused functional deficits.

Results: Mice harboring OVA-expressing B16 melanoma that werepre-treated for 3 days with compound 25 before adoptive transfer ofactivated OVA-specific OT1 T cells showed an unexpected improvement intumor control and survival. See FIGS. 50A-D.

Example 16 Compound 25 Inhibits 3LL Tumor Growth

5×10⁶ 3 LL cells were implanted subcutaneously into the right flank inC57BL/6J male mice. 3LL tumor-bearing mice were treated with compound 25(1 mg/kg) starting at day 7 after tumor inoculation. After 7 days, alower dose (0.3 mg/kg) of compound 25 was used. FIG. 51A shows thatcompound 25 reduced tumor growth. FIG. 51B shows that percentages oflive Ly6c lo Ly6G Hi, Ly6c hi Grl, CD8+, and CD4+ of cells from blood in3LL tumor bearing mice, analyzed by flow cytometry at the indicated timepoint. FIGS. 51C-D demonstrate an increased ratio of CD8 cells to MDSCs+and TANs from blood and tumor-infiltrating leukocytes (TIL).

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All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art. In case of aconflict between the specification and any of the incorporatedreferences, the specification (including any amendments thereof, whichmay be based on an incorporated reference), shall control. Standardart-accepted meanings of terms are used herein unless indicatedotherwise. Standard abbreviations for various terms are used herein.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

1.-21. (canceled)
 22. A method of treating cancer in a subject in needthereof, the method comprising sequentially administering to thesubject: i. a therapeutically effective amount of an immunotherapeuticagent; and ii. a therapeutically effective amount of compound havingformula (IIA):

wherein: R₁ is selected from the group consisting of H and C1-6 alkyl;R₁₁ is selected from the group consisting of H, methyl, isopropyl,sec-butyl, CH₂CH(CH₃)₂, benzyl, p-hydroxybenzyl CH₂OH, CH(OH)CH₃,CH₂-3-indoyl, CH₂COOH, CH₂CH₂COOH, —CH₂C(O)NH₂, CH₂CH₂C(O)NH₂, CH₂SH,CH₂CH₂SCH₃, (CH₂)₄NH₂, (CH₂)₃NHC(═NH)NH₂, and CH₂-3-imidazoyl; R₁₂ isselected from the group consisting of H, C₁₋₄ alkyl, and —C(═O)R₁₃; andR₁₃ is C₁₋₄ alkyl.
 23. The method of claim 22, wherein the compoundhaving Formula (IIA) is:


24. The method of claim 23, wherein the immunotherapeutic agent is animmune checkpoint blockade therapy.
 25. The method of claim 24, whereinthe immune checkpoint blockade therapy is a PD-1 antagonist, a PD-L1antagonist, a CTLA-4 antagonist, a LAG3 antagonist, or a B7-H₃antagonist, or a combination thereof.
 26. The method of claim 25,wherein the immune checkpoint blockade therapy is a PD-1 antagonist. 27.The method of claim 26, wherein the PD-1 antagonist is atezolizumab,nivolumab, pembrolizumab, or pidilizumab.
 28. The method of claim 25,wherein the immune checkpoint blockade therapy is a PD-L1 antagonist.29. The method of claim 28, wherein the PD-L1 antagonist is BMS-936559,MEDI4736, or MSB0010718C.
 30. The method of claim 25, wherein the immunecheckpoint blockade therapy is a CTLA-4 antagonist.
 31. The method ofclaim 30, wherein the CTLA-4 antagonist is ipilimumab or tremelimumab.32. The method of claim 25, wherein the immune checkpoint blockadetherapy is a LAG3 antagonist.
 33. The method of claim 32, wherein theLAG3 antagonist is BMS-986016 or IMP321.
 34. The method of claim 25,wherein the immune checkpoint blockade therapy is a B7-H₃ antagonist.35. The method of claim 34, wherein the B7-H₃ antagonist is MGA271. 36.The method of claim 24, wherein the cancer is refractory tomonotreatment with the immunotherapeutic agent.
 37. The method of claim24, wherein the cancer is a solid tumor.
 38. The method of claim 24,wherein the cancer is non-small cell lung cancer.
 39. The method ofclaim 24, wherein the cancer is head and neck squamous cell carcinoma.40. The method of claim 24, wherein the cancer is celnasopharyngealcancer, synovial cancer, hepatocellular cancer, renal cancer, cancer ofconnective tissues, melanoma, lung cancer, bowel cancer, colon cancer,rectal cancer, colorectal cancer, brain cancer, throat cancer, oralcancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma,gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma,neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenalcancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer,oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bonecancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer ofunknown primary site, carcinoid, carcinoid of gastrointestinal tract,fibrosarcoma, breast cancer, Paget's disease, cervical cancer,colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer,head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, livercancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicularcancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skincancer, mesothelioma, multiple myeloma, ovarian cancer, endocrinepancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer,penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma,small intestine cancer, stomach cancer, thymus cancer, thyroid cancer,trophoblastic cancer, hydatidiform mole, uterine cancer, endometrialcancer, vagina cancer, vulva cancer, acoustic neuroma, mycosisfungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer,heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer,palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer,pleural cancer, salivary gland cancer, tongue cancer, or tonsil cancer.41. A method of treating colorectal cancer, melanoma, lung cancer,hepatocellular carcinoma, head and neck cancer, breast cancer, esophaguscancer, Hodgkin's disease, bladder cancer, or renal cell carcinoma in asubject in need thereof, the method comprising sequentiallyadministering to the subject: (i) a therapeutically effective amount ofatezolizumab, nivolumab, pembrolizumab, pidilizumab, ipilimumab,tremelimumab, MEDI4736, or MSB0010718C, or a combination thereof; and(ii) a therapeutically effective amount of: